Novel telomerase

ABSTRACT

The present invention is directed to novel telomerase nucleic acids and amino acids. In particular, the present invention is directed to nucleic acid and amino acid sequences encoding various telomerase protein subunits and motifs, including the 123 kDa and 43 kDa telomerase protein subunits of  Euplotes aediculatus , and related sequences from Schizosaccharomyces, Saccharomyces sequences, and human telomerase. The present invention is also directed to polypeptides comprising these telomerase protein subunits, as well as functional polypeptides and ribonucleoproteins that contain these subunits.

[0001] The present application is a Continuation-In-Part application ofU.S. patent application Ser. No. 08/______, filed May 6, 1997, which isa Continuation-In-Part application of U.S. patent application Ser. No.08/______,filed Apr. 25, 1997, which is a Continuation-in-Part of U.S.patent application Ser. No. 08/______, filed Apr. 18, 1997, which is aContinuation-in-Part of U.S. patent application Ser. No. 08/724,643,filed on Oct. 1, 1996.

FIELD OF THE INVENTION

[0002] The present invention is related to novel telomerase genes andproteins. In particular, the present invention is directed to atelomerase isolated from Euplotes aediculatus, the two polypeptidesubunits of this telomerase, as well as sequences of theSchizosaccharomyces, Tetrahymena, and human homologs of the E.aediculatus telomerase.

BACKGROUND OF THE INVENTION

[0003] Telomeres, the protein-DNA structures physically located on theends of the eukaryotic organisms, are required for chromosome stabilityand are involved in chromosomal organization within the nucleus (Seee.g., Zakian, Science 270:1601 [1995]; Blackburn and Gall, J. Mol.Biol., 120:33 [1978]; Oka et al., Gene 10:301 [1980]; and Klobutcher etal., Proc. Natl. Acad. Sci., 78:3015 [1981]). Telomeres are believed tobe essential in such organisms as yeasts and probably most othereukaryotes, as they allow cells to distinguish intact from brokenchromosomes, protect chromosomes from degradation, and act as substratesfor novel replication mechanisms. Telomeres are generally replicated ina complex, cell cycle and developmentally regulated, manner by“telomerase,” a telomere-specific DNA polymerase. However,telomerase-independent means for telomere maintenance have beendescribed. In recent years, much attention has been focused ontelomeres, as telomere loss has been associated with chromosomal changessuch as those that occur in cancer and aging.

[0004] Telomeric DNA

[0005] In most organisms, telomeric DNA has been reported to consist ofa tandem array of very simple sequences, which in many cases are shortand precise. Typically, telomeres consist of simple repetitive sequencesrich in G residues in the strand that runs 5′ to 3′ toward thechromosomal end. For example, telomeric DNA in Tetrahymena is comprisedof sequence T₂G₄, while in Oxytricha, the sequence is T₄G₄, and inhumans the sequence is T₂AG₃ (See e.g., Zakian, Science 270:1601 [1995];and Lingner et al., Genes Develop., 8:1984 [1994]). However,heterogenous telomeric sequences have been reported in some organisms(e.g., the sequence TG₁₋₃ in Saccharomyces). In addition, the repeatedtelomeric sequence in some organisms is much longer, such as the 25 basepair sequence of Kluyveromyces lactis. Moreover, the telomeric structureof some organisms is completely different. For example, the telomeres ofDrosophila are comprised of a transposable element (See, Biessman etal., Cell 61:663 [1990]; and F.-m Sheen and Levis, Proc. Natl. Acad.Sci., 91:12510 [1994]).

[0006] The telomeric DNA sequences of many organisms have beendetermined (See e.g., Zakian, Science 270:1601 [1995]). However, it hasbeen noted that as more telomeric sequences become known, it is becomingincreasingly difficult to identify even a loose consensus sequence todescribe them (Zakian, supra). Furthermore, it is known that the averageamount of telomeric DNA varies between organisms. For example, mice mayhave as many as 150 kb (kilobases) of telomeric DNA per telomere, whilethe telomeres of Oxytricha macronuclear DNA molecules are only 20 bp inlength (Kipling and Cooke, Nature 347:400 [1990]; Starling et al.,Nucleic Acids Res., 18:6881 [1990]; and Klobutcher et al., Proc. Natl.Acad. Sci., 78:3015 [1981]). Moreover, in most organisms, the amount oftelomeric DNA fluctuates. For example, the amount of telomeric DNA atindividual yeast telomeres in a wild-type strain may range fromapproximately 200 to 400 bp, with this amount of DNA increasing anddecreasing stoichastically (Shampay and Blackburn, Proc. Natl. Acad.Sci., 85:534 [1988]). Heterogeneity and spontaneous changes in telomerelength may reflect a complex balance between the processes involved indegradation and lengthening of telomeric tracts. In addition, genetic,nutritional and other factors may cause increases or decreases intelomeric length (Lustig and Petes, Natl. Acad. Sci., 83:1398 [1986];and Sandell et al., Cell 91:12061 [1994]). The inherent heterogeneity ofvirtually all telomeric DNAs suggests that telomeres are not maintainedvia conventional replicative processes.

[0007] In addition to the telomeres themselves, the regions locatedadjacent to telomeres have been studied. For example, in most organisms,the sub-telomeric regions immediately internal to the simple repeatsconsist of middle repetitive sequences, designated astelomere-associated (“TA”) DNA. These regions bear some similarity withthe transposon telomeres of Drosophila. In Saccharomyces, two classes ofTA elements, designated as “X” and “Y,”′ have been described (Chan andTye, Cell 33:563 [1983]). These elements may be found alone or incombination on most or all telomeres.

[0008] Telomeric Structural Proteins

[0009] Various structural proteins that interact with telomeric DNA havebeen described which are distinct from the protein components of thetelomerase enzyme. Such structural proteins comprise the “telosome” ofSaccharomyces chromosomes (Wright et al., Genes Develop., 6:197 [1992])and of ciliate macronuclear DNA molecules (Gottschling and Cech, Cell38:501 [1984]; and Blackburn and Chiou, Proc. Natl. Acad. Sci., 78:2263[1981]). The telosome is a non-nucleosomal, but discrete chromatinstructure that encompasses the entire terminal array of telomericrepeats. In Saccharomyces, the DNA adjacent to the telosome is packagedinto nucleosomes. However, these nucleosomes are reported to differ fromthose in most other regions of the yeast genome, as they have featuresthat are characteristic of transcriptionally inactive chromatin (Wrightet al., Genes Develop., 6:197 [1992]; and Braunstein et al., GenesDevelop., 7:592 [1993]). In mammals, most of the simple repeatedtelomeric DNA is packaged in closely spaced nucleosomes (Makarov et al.,Cell 73:775 [1993]; and Tommerup et al., Mol. Cell. Biol., 14:5777[1994]). However, the telomeric repeats located at the very ends of thehuman chromosomes are found in a telosome-like structure.

[0010] Telomere Replication

[0011] Complete replication of the ends of linear eukaryotic chromosomespresents special problems for conventional methods of DNA replication.For example, conventional DNA polymerases cannot begin DNA synthesis denovo, rather, they require RNA primers which are later removed duringreplication. In the case of telomeres, removal of the RNA primer fromthe lagging-strand end would necessarily leave a 5′-terminal gap,resulting in the loss of sequence if the parental telomere wasblunt-ended (Watson, Nature New Biol., 239:197 [1972]; Olovnikov, J.Theor. Biol., 41:181 [1973]). However, the described telomeres have 3′overhangs (Klobutcher et al., Proc. Natl. Acad. Sci., 58:3015 [1981];Henderson and Blackburn, Mol. Cell. Biol., 9:345 [1989]; and Wellingeret al., Cell 72:51 [1993]). For these molecules, it is possible thatremoval of the lagging-strand 5′-terminal RNA primer could regeneratethe 3′ overhang without loss of sequence on this side of the molecule.However, loss of sequence information on the leading-strand end wouldoccur, because of the lack of a complementary strand to act as templatein the synthesis of a 3′ overhang (Zahler and Prescott, Nucleic AcidsRes., 16:6953 [1988]; Lingner et al., Science 269:1533 [1995]).

[0012] Nonetheless, complete replication of the chromosomes must occur.While conventional DNA polymerases cannot accurately reproducechromosomal DNA ends, specialized factors exist to ensure their completereplication. Telomerase is a key component in this process. Telomeraseis a ribonucleoprotein (RNP) particle and polymerase that uses a portionof its internal RNA moiety as a template for telomere repeat DNAsynthesis (Yu et al., Nature 344:126 [1990]; Singer and Gottschling,Science 266:404 [1994]; Autexier and Greider, Genes Develop., 8:563[1994]; Gilley et al., Genes Develop., 9:2214 [1995]; McEachern andBlackburn, Nature 367:403 [1995]; Blackburn, Ann. Rev. Biochem., 61:113[1992];. Greider, Ann. Rev. Biochem., 65:337 [1996]). The activity ofthis enzyme depends upon both its RNA and protein components tocircumvent the problems presented by end replication by using RNA (i.e.,as opposed to DNA) to template the synthesis of telomeric DNA.

[0013] Telomerases extend the G strand of telomeric DNA. A combinationof factors, including telomerase processivity, frequency of action atindividual telomeres, and the rate of degradation of telomeric DNA,contribute to the size of the telomeres (i.e., whether they arelengthened, shortened, or maintained at a certain size). In vitro,telomerases may be extremely processive, with the Tetrahymena telomeraseadding an average of approximately 500 bases to the G strand primerbefore dissociation of the enzyme (Greider, Mol. Cell. Biol., 114572[1991]).

[0014] Importantly, telomere replication is regulated both bydevelopmental and cell cycle factors. It has been hypothesized thataspects of telomere replication may act as signals in the cell cycle.For example, certain DNA structures or DNA-protein complex formationsmay act as a checkpoint to indicate that chromosomal replication hasbeen completed (See e.g., Wellinger et al., Mol. Cell. Biol., 13:4057[1993]). In addition, it has been observed that in humans, telomeraseactivity is not detectable in most somatic tissues, although it isdetected in many tumors (Wellinger, supra). This telomere length mayserve as a mitotic clock, which serves to limit the replicationpotential of cells in vivo and/or in vitro. What remains needed in theart is a method to study the role of telomeres and their replication innormal as well as abnormal cells (i.e., cancerous cells). Anunderstanding of telomerase and its function is needed in order todevelop means for use of telomerase as a target for cancer therapy oranti-aging processes.

SUMMARY OF THE INVENTION

[0015] The present invention provides compositions and methods forpurification and use of telomerase. In particular, the present inventionis directed to telomerase and co-purifying polypeptides obtained fromEuplotes aediculatus, as well as other organisms (e.g.,Schizosaccharomyces, Tetrahymena, and humans). The present inventionalso provides methods useful for the detection and identification oftelomerase homologs in other species and genera of organisms.

[0016] The present invention provides heretofore unknown telomerasesubunit proteins of E. aediculatus of approximately 123 kDa and 43 kDa,as measured on SDS-PAGE. In particular, the present invention providessubstantially purified 123 kDa and 43 kDa telomerase protein subunits.

[0017] One aspect of the invention features isolated and substantiallypurified polynucleotides which encode telomerase subunits (i.e., the 123kDa and 43 kDa protein subunits). In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:1, or variantsthereof. In an alternative embodiment, the present invention providesfragments of the isolated (i.e., substantially purified) polynucleotideencoding the telomerase 123 kDa subunit of at least 10 amino acidresidues in length. The invention further contemplates fragments of thispolynucleotide sequence (i.e., SEQ ID NO: 1) that are at least 6nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least100 nucleotides, at least 250 nucleotides, and at least 500 nucleotidesin length. In addition, the invention features polynucleotide sequencesthat hybridize under stringent conditions to SEQ ID NO:1, or fragmentsthereof. The present invention further contemplates a polynucleotidesequence comprising the complement of the nucleic acid of SEQ ID NO:1,or variants thereof.

[0018] The present invention also provides the polynucleotide with thesequence of SEQ ID NO:3. In particular, the present invention providesthe polynucleotide sequence comprising at least a portion of the nucleicacid sequence of SEQ ID NO:3, or variants, thereof. In one embodiment,the present invention provides fragments of the isolated (i.e.,substantially purified) polynucleotide encoding the telomerase 43 kDasubunit of at least 10 amino acid residues in length. The invention alsoprovides an isolated polynucleotide sequence encoding the polypeptide ofSEQ ID NOS:4-6, or variants thereof. The invention further contemplatesfragments of this polynucleotide sequence (i.e., SEQ ID NO:3) that areat least 5 nucleotides, at least 20 nucleotides, at least 100nucleotides, at least 250 nucleotides, and at least 500 nucleotides inlength. In addition, the invention features polynucleotide sequencesthat hybridize under stringent conditions to SEQ ID NO:3, or fragmentsthereof. The present invention further contemplates a polynucleotidesequence comprising the complement of the nucleic acid of SEQ ID NO:3,or variants thereof.

[0019] The present invention provides a substantially purifiedpolypeptide comprising at least a portion of the amino acid sequence ofSEQ ID NO:2, or variants thereof. In one embodiment, the portion of thepolypeptide sequence comprises fragments of SEQ ID NO:2, having a lengthgreater than 10 amino acids. However, the invention also contemplatespolypeptide sequences of various lengths, the sequences of which areincluded within SEQ ID NO:2, ranging from 5-500 amino acids. The presentinvention also provides an isolated polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2, or variants, thereof.

[0020] The present invention provides a substantially purifiedpolypeptide comprising at least a portion of the amino acid sequenceselected from the group consisting of SEQ ID NO:4-6, or variantsthereof. In one embodiment, the portion of the polypeptide comprisesfragments of SEQ ID NO:4, having a length greater than 10 amino acids.In an alternative embodiment, the portion of the polypeptide comprisesfragments of SEQ ID NO:5, having a length greater than 10 amino acids.In yet another alternative embodiment, the portion of the polypeptidecomprises fragments of SEQ ID NO:6, having a length greater than 10amino acids. The present invention also contemplates polypeptidesequences of various lengths, the sequences of which are included withinSEQ ID NOS:4, 5, and/or 6, ranging from 5 to 500 amino acids.

[0021] The present invention also provides a telomerase complexcomprised of at least one purified 123 kDa telomerase protein subunit,at least one a purified 43 kDa telomerase protein subunit, and purifiedRNA. In a preferred embodiment, the telomerase complex comprises onepurified 123 kDa telomerase protein subunit, one purified 43 kDatelomerase protein subunit, and purified telomerase RNA. In onepreferred embodiment, the telomerase complex comprises an 123 kDa and/ortelomerase protein subunit obtained from Euplotes aediculatus. It iscontemplated that the 123 kDa telomerase protein subunit of thetelomerase complex be encoded by SEQ ID NO:1. It is also contemplatedthat the 123 kDa telomerase protein subunit of the telomerase complex becomprised of SEQ ID NO:2. It is also contemplated that the 43 kDatelomerase protein subunit of the telomerase complex be obtained fromEuplotes aediculatus. It is further contemplated that the 43 kDatelomerase subunit of the telomerase complex be encoded by SEQ ID NO:3.It is also contemplated that the 43 kDa telomerase protein subunit ofthe telomerase complex be comprised of the amino acid sequence selectedfrom the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.It is contemplated that the purified RNA of the telomerase complex becomprised of the RNA encoded by such sequences as those disclosed byLinger et al., (Lingner et al., Genes Develop., 8:1985 [1994]). In apreferred embodiment, the telomerase complex is capable of replicatingtelomeric DNA.

[0022] The present invention also provides methods for identifyingtelomerase protein subunits in eukaryotic organisms other than E.aediculatus . These methods are comprised of multiple steps. The firststep is the synthesis of at least one probe or primer oligonucleotidethat encodes at least a portion of the amino acid sequence of SEQ IDNOS:2, 4, 5, or 6. In the alternative, the synthesized probe or primeroligonucleotides are complementary to at least a portion of the aminoacid sequence of SEQ ID NO:2, 4, 5, or 6. The next step comprisesexposing at least one of the probe or primer oligonucleotide(s) tonucleic acid comprising the genome or, in the alternative, the expressedportion of the genome of the other organism (i.e., the non-E.aediculatus organism), under conditions suitable for the formation ofnucleic acid hybrids. Next, the hybrids are identified with or withoutamplification, using a DNA polymerase (e.g., Taq, or any other suitablepolymerase known in the art). Finally, the sequence of the hybrids aredetermined using methods known in the art, and the sequences of thederived amino acid sequences analyzed for their similarity to SEQ IDNOS:2, 4, 5, or 6.

[0023] The present invention also provides methods for identifyingnucleic acid sequences encoding telomerase protein subunits ineukaryotic organisms comprising the steps of: providing a samplesuspected of containing nucleic acid encoding an eukaryotic telomeraseprotein subunit; at least one oligonucleotide primer complementary tothe nucleic acid sequence encoding at least a region of an Euplotesaediculatus telomerase protein subunit; and iii) a polymerase; exposingthe sample to the at least one oligonucleotide primer and the polymeraseunder conditions such that the nucleic acid encoding the eukaryotictelomerase protein subunit is amplified; determining the sequence of theeukaryotic telomerase protein subunit; and comparing the sequence of theeukaryotic telomerase protein subunit and the Euplotes aediculatustelomerase protein subunit. In one preferred embodiment, the Euplotesaediculatus telomerase subunit comprises at least a portion of SEQ IDNO: 1. In an alternative preferred embodiment, the Euplotes aediculatustelomerase subunit comprises at least a portion of SEQ ID NO:3.

[0024] Thus, the present invention also provides methods foridentification of telomerase protein subunits in eukaryotic organismsother than E. aediculatus . In addition, the present invention providesmethods for comparisons between the amino acid sequences of SEQ IDNOS:2, 4, 5, or 6, and the amino acid sequences derived from genesequences of other organisms or obtained by direct amino acid sequenceanalysis of protein. The amino acid sequences shown to have the greatestdegree of identity (i.e., homology) to SEQ ID NOS:2, 4, 5, or 6, maythen selected for further testing. Sequences of particular importanceare those that share identity with the reverse transcriptase motif ofthe Euplotes sequence. Once identified, the proteins with the sequencesshowing the greatest degree of identity may be tested for their role intelomerase activity by genetic or biochemical methods, including themethods set forth in the Examples below.

[0025] The present invention also provides methods for purification oftelomerase comprising the steps of providing a sample containingtelomerase, an affinity oligonucleotide, a displacement oligonucleotide;exposing the sample to the affinity oligonucleotide under conditionswherein the affinity oligonucleotide binds to the telomerase to form atelomerase-oligonucleotide complex; and exposing theoligonucleotide-telomerase complex to the displacement oligonucleotideunder conditions such that the telomerase is released from the template.In a preferred embodiment, the method comprises the further step ofeluting the telomerase. In another preferred embodiment, the affinityoligonucleotide comprises an antisense portion and a biotin residue. Itis contemplated that during the exposing step, the biotin residue of theaffinity oligonucleotide binds to an avidin bead and the antisenseportion binds to the telomerase. It is also contemplated that during theexposing step, the displacement oligonucleotide binds to the affinityoligonucleotide.

[0026] The present invention further provides substantially purifiedpolypeptides comprising the amino acid sequence comprising SEQ IDNOS:61, 63, 64, 65, 67, and 68. In another embodiment, the presentinvention also provides purified, isolated polynucleotide sequencesencoding the polypeptides comprising the amino acid sequences of SEQ IDNOS:61, 63, 64, 65, 67, and 68. The present invention contemplatesportions or fragments of SEQ ID NOS:61, 63, 64, 65, 67, and 68, ofvarious lengths. In one embodiment, the portion of polypeptide comprisesfragments of lengths greater than 10 amino acids. However, the presentinvention also contemplates polypeptide sequences of various lengths,the sequences of which are included within SEQ ID NOS:61, 63, 64, 65,67, and 68, ranging from 5 to 500 amino acids (as appropriate, based onthe length of SEQ ID NOS:61, 63, 64, 65, 67, and 68).

[0027] The present invention also provides nucleic acid sequencescomprising SEQ ID NOS:55, 62, 66, and 69, or variants thereof. Thepresent invention further provides fragments of the isolatedpolynucleotide sequences that are at least 6 nucleotides, at least 25nucleotides, at least 30 nucleotides, at least 50 nucleotides, at least100 nucleotides, at least 250 nucleotides, and at least 500 nucleotidesin length (as appropriate for the length of the sequence of SEQ IDNOS:55, 62, 66, and 69, or variants thereof).

[0028] In particularly preferred embodiments, the polynucleotidehybridizes specifically to telomerase sequences, wherein the telomerasesequences are selected from the group consisting of human, Euplotesaediculatus, Oxytricha, Schizosaccharomyces, and Saccharomycestelomerase sequences. In other preferred embodiments, the presentinvention provides polynucleotide sequences comprising the complement ofnucleic acid sequences selected from the group consisting of SEQ IDNOS:55, 62, 66, and 69, or variants thereof. In yet other preferredembodiments, the present invention provides polynucleic acid sequencesthat hybridize under stringent conditions to at least one nucleic acidsequence selected from the group consisting of SEQ ID NO:55, 62, 66, and69. In a further embodiment, the polynucleotide sequence comprises apurified, synthetic nucleotide sequence having a length of about ten tothirty nucleotides.

[0029] In alternative preferred embodiments, the present inventionprovides polynucleotide sequences corresponding to the human telomerase,including SEQ ID NOS:113 and 117, and their complementary sequences. Theinvention further contemplates fragments of these polynucleotidesequence (i.e., SEQ ID NOS: 113, and 117) that are at least 5nucleotides, at least 20 nucleotides, at least 100 nucleotides, at least250 nucleotides, and at least 500 nucleotides in length. The inventionfurther contemplates fragments of the complements of thesepolynucleotide sequences (i.e., SEQ ID NOS: 113, and 117) that are atleast 5 nucleotides, at least 20 nucleotides, at least 100 nucleotides,at least 250 nucleotides, and at least 500 nucleotides in length. Inaddition, the invention features polynucleotide sequences that hybridizeunder stringent conditions to SEQ ID NOS:113 and 117, and/or fragments,and/or the complementary sequences thereof. The present inventionfurther contemplates a polynucleotide sequence comprising the complementof the nucleic acids of SEQ ID NOS:113 and 117, or variants thereof. Ina further embodiment, the polynucleotide sequence comprises a purified,synthetic nucleotide sequence corresponding to a fragment of SEQ IDNOS:113 and/or 117, having a length of about ten to thirty nucleotides.The present invention further provides plasmid pGRN121 (ATCC accession##20916), and the lambda clone 25-1.1 (ATCC accession #______).

[0030] The present invention further provides substantially purifiedpolypeptides comprising the amino acid sequence comprising SEQ IDNOS:114-116, and 118. In another embodiment, the present invention alsoprovides purified, isolated polynucleotide sequences encoding thepolypeptides comprising the amino acid sequences of SEQ ID NOS:114-116,and 118. The present invention contemplates portions or fragments of SEQID NOS:114-116, and 118, of various lengths. In one embodiment, theportion of polypeptide comprises fragments of lengths greater than 10amino acids. However, the present invention also contemplatespolypeptide sequences of various lengths, the sequences of which areincluded within SEQ ID NOS:114-116, and 118, ranging from 5 to 1100amino acids (as appropriate, based on the length of SEQ ID NOS:114-116,and 118).

[0031] The present invention also provides methods for detecting thepresence of nucleotide sequences encoding at least a portion of humantelomerase in a biological sample, comprising the steps of, providing: abiological sample suspected of containing nucleic acid corresponding tothe nucleotide sequence set forth in SEQ ID NO:62; the nucleotide of SEQID NO:62 or fragment(s) thereof; combining the biological sample withthe nucleotide under conditions such that a hybridization complex isformed between the nucleic acid and the nucleotide; and detecting thehybridization complex.

[0032] In one embodiment of the method the nucleic acid corresponding tothe nucleotide sequence of SEQ ID NO:62, is ribonucleic acid, while inan alternative embodiment, the nucleotide sequence is deoxyribonucleicacid. In yet another embodiment of the method the detected hybridizationcomplex correlates with expression of the polynucleotide of SEQ IDNO:62, in the biological sample. In yet another embodiment of themethod, detection of the hybridization complex comprises conditions thatpermit the detection of alterations in the polynucleotide of SEQ IDNO:62 in the biological sample.

[0033] The present invention also provides antisense moleculescomprising the nucleic acid sequence complementary to at least a portionof the polynucleotide of SEQ ID NO:55, 62, 66, 67, and 69. In analternatively preferred embodiment, the present invention also providespharmaceutical compositions comprising antisense molecules of SEQ IDNOS:55, 62, 67, and 69, and a pharmaceutically acceptable excipientand/or other compound (e.g., adjuvant).

[0034] In yet another embodiment, the present invention providespolynucleotide sequences contained on recombinant expression vectors. Inone embodiment, the expression vector containing the polynucleotidesequence is contained within a host cell.

[0035] The present invention also provides methods for producingpolypeptides comprising the amino acid sequence of SEQ ID NOS:61, 63,65, 67, or 69, the method comprising the steps of: culturing a host cellunder conditions suitable for the expression of the polypeptide; andrecovering the polypeptide from the host cell culture.

[0036] The present invention also provides purified antibodies thatbinds specifically to a polypeptide comprising at least a portion of theamino acid sequence of SEQ ID NOS:55, 61, 63, 64, 65, 67, and/or 68. Inone embodiment, the present invention provides a pharmaceuticalcomposition comprising at least one antibody, and a pharmaceuticallyacceptable excipient.

[0037] The present invention further provides methods for the detectionof human telomerase in a biological sample comprising the steps of:providing a biological sample suspected of expressing human telomeraseprotein; and at least one antibody that binds specifically to at least aportion of the amino acid sequence of SEQ ID NOS:55, 61, 63, 64, 65, 67,and/or 68; combining the biological sample and antibody(ies) underconditions such that an antibody:protein complex is formed; anddetecting the complex wherein the presence of the complex correlateswith the expression of the protein in the biological sample.

[0038] The present invention further provides substantially purifiedpeptides comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOS:71, 73, 75, 77, 79, 82, 83, 83, 85, 86, and101. In an alternative embodiment, the present invention providespurified, isolated polynucleotide sequences encoding the polypeptidecorresponding to these sequences. In preferred embodiments, thepolynucleotide hybridizes specifically to telomerase sequences, whereinthe telomerase sequences are selected from the group consisting ofhuman, Euplotes aediculatus, Oxytricha, Schizosaccharomyces,Saccharomyces and Tetrahymena telomerase sequences. In yet anotherembodiment, the polynucleotide sequence comprises the complement of anucleic acid sequence selected from the group consisting of SEQ IDNOS:70, 72, 74, 76, 78, 80, 81, 100, 113, 117, and variants thereof. Ina further embodiment, the polynucleotide sequence that hybridizes understringent conditions to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS:66, 69, 80, and 81. In yet another embodiment,the polynucleotide sequence is selected from the group consisting of SEQID NOS:70, 72, 74, 76, 78, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, and 117.In an alternative embodiment, the nucleotide sequence comprises apurified, synthetic nucleotide sequence having a length of about ten tofifty nucleotides.

[0039] The present invention also provides methods for detecting thepresence of nucleotide sequences encoding at least a portion of humantelomerase in a biological sample, comprising the steps of, providing: abiological sample suspected of containing nucleic acid corresponding tothe nucleotide sequence of SEQ ID NO:100, and/or SEQ ID NO:113, and/orSEQ ID NO:117; the nucleotide of SEQ ID NO:100, and/or SEQ ID NO:113,and/or SEQ ID NO:117, or fragment(s) thereof; combining the biologicalsample with the nucleotide under conditions such that a hybridizationcomplex is formed between the nucleic acid and the nucleotide; anddetecting the hybridization complex.

[0040] In one embodiment of the method the nucleic acid corresponding tothe nucleotide sequence of SEQ ID NO:100, and/or SEQ ID NO:113, and/orSEQ ID NO:117, is ribonucleic acid, while in an alternative embodiment,the nucleotide sequence is deoxyribonucleic acid. In yet anotherembodiment of the method the detected hybridization complex correlateswith expression of the polynucleotide of SEQ ID NO:100, and/or SEQ IDNO:113, and/or SEQ ID NO:117, in the biological sample. In yet anotherembodiment of the method, detection of the hybridization complexcomprises conditions that permit the detection of alterations in thepolynucleotide of SEQ ID NO: 100 and/or SEQ ID NO: 113, and/or SEQ IDNO: 117, in the biological sample.

[0041] The present invention also provides antisense moleculescomprising the nucleic acid sequence complementary to at least a portionof the polynucleotide of SEQ ID NO:82, 100, 113, and 117. In analternatively preferred embodiment, the present invention also providespharmaceutical compositions comprising antisense molecules of SEQ IDNOS:82, 100, 113, 117, and a pharmaceutically acceptable excipientand/or other compound (e.g., adjuvant).

[0042] In yet another embodiment, the present invention providespolynucleotide sequences contained on recombinant expression vectors. Inone embodiment, the expression vector containing the polynucleotidesequence is contained within a host cell.

[0043] The present invention also provides methods for producingpolypeptides comprising the amino acid sequence of SEQ ID NOS:82, 83,84, 85, 86, 101, 114, 115, 116, and/or 118, the method comprising thesteps of: culturing a host cell under conditions suitable for theexpression of the polypeptide; and recovering the polypeptide from thehost cell culture.

[0044] The present invention also provides purified antibodies thatbinds specifically to a polypeptide comprising at least a portion of theamino acid sequence of SEQ ID NOS:71, 73, 75, 77, 79, 82, 83, 84, 85,86, 101, 114, 115, 116, and/or 118. In one embodiment, the presentinvention provides a pharmaceutical composition comprising at least oneantibody, and a pharmaceutically acceptable excipient.

[0045] The present invention further provides methods for the detectionof human telomerase in a biological sample comprising the steps of:providing a biological sample suspected of expressing human telomeraseprotein; and at least one antibody that binds specifically to at least aportion of the amino acid sequence of SEQ ID NOS:71, 73, 75, 77, 79, 82,83, 84, 85, 86, 87, 101, 114, 115, 116, and/or 118, combining thebiological sample and antibody(ies) under conditions such that anantibody:protein complex is formed; and detecting the complex whereinthe presence of the complex correlates with the expression of theprotein in the biological sample.

DESCRIPTION OF THE FIGURES

[0046]FIG. 1 is a schematic diagram of the affinity purification oftelomerase showing the binding and displacement elution steps.

[0047]FIG. 2 is a photograph of a Northern blot of telomerasepreparations obtained during the purification protocol.

[0048]FIG. 3 shows telomerase activity through the purificationprotocol.

[0049]FIG. 4 is a photograph of a SDS-PAGE gel, showing the presence ofan approximately 123 kDa polypeptide and an approximately 43 kDadoublet.

[0050]FIG. 5 is a graph showing the sedimentation coefficient oftelomerase.

[0051]FIG. 6 is a photograph of a polyacrylamide/urea gel with 36%formamide.

[0052]FIG. 7 shows the putative alignments of telomerase RNA template,with SEQ ID NOS:43 and 44 in Panel A, and SEQ ID NOS:45 and 46 in PanelB.

[0053]FIG. 8 is a photograph of lanes 25-30 of the gel shown in FIG. 6,shown at a lighter exposure level.

[0054]FIG. 9 shows the DNA sequence of the gene encoding the 123 kDatelomerase protein subunit (SEQ ID NO:1).

[0055]FIG. 10 shows the amino acid sequence of the 123 kDa telomeraseprotein subunit (SEQ ID NO:2).

[0056]FIG. 11 shows the DNA sequence of the gene encoding the 43 kDatelomerase protein subunit (SEQ ID NO:3).

[0057]FIG. 12 shows the DNA sequence, as well as the amino acidsequences of all three open reading frames of the 43 kDa telomeraseprotein subunit (SEQ ID NOS:4-6).

[0058]FIG. 13 shows a sequence comparison between the 123 kDa telomeraseprotein subunit of E. aediculatus (SEQ ID NO:2) and the 80 kDapolypeptide subunit of T. thermophila (SEQ ID NO:52).

[0059]FIG. 14 shows a sequence comparison between the 123 kDa telomeraseprotein subunit of E. aediculatus (SEQ ID NO:2) and the 95 kDatelomerase polypeptide of T. thermophila (SEQ ID NO:54).

[0060]FIG. 15 shows the best-fit alignment between a portion of the“La-domain” of the 43 kDa telomerase protein subunit of E. aediculatus(SEQ ID NO:9) and a portion of the 95 kDa polypeptide subunit of T.thermophila (SEQ ID NO: 10).

[0061]FIG. 16 shows the best-fit alignment between a portion of the“La-domain” of the 43 kDa telomerase protein subunit of E. aediculatus(SEQ ID NO:11) and a portion of the 80 kDa polypeptide subunit of Tthermophila (SEQ ID NO: 12).

[0062]FIG. 17 shows the alignment and motifs of the polymerase domain ofthe 123 kDa telomerase protein subunit of E. aediculatus (SEQ ID NOS:13and 18) and the polymerase domains of various reverse transcriptases(SEQ ID NOS:14-17, and 19-22).

[0063]FIG. 18 shows the alignment of a domain of the 43 kDa telomeraseprotein subunit (SEQ ID NO:23) with various La proteins (SEQ IDNOS:24-27).

[0064]FIG. 19 shows the nucleotide sequence encoding the T. thermophila80 kDa protein subunit (SEQ ID NO:51).

[0065]FIG. 20 shows the amino acid sequence of the T thermophila 80 kDaprotein subunit (SEQ ID NO:52).

[0066]FIG. 21 shows the nucleotide sequence encoding the T. thermophila95 kDa protein subunit (SEQ ID NO:53).

[0067]FIG. 22 shows the amino acid sequence of the T, thermophila 95 kDaprotein subunit (SEQ ID NO:54).

[0068]FIG. 23 shows the amino acid sequence of L8543.12 (“Est2p”) (SEQID NO:55).

[0069]FIG. 24 shows the alignment of the Oxytricha PCR product (SEQ IDNO:58) with the Euplotes sequence (SEQ ID NO:59).

[0070]FIG. 25 shows the alignment of the human telomere amino acidmotifs (SEQ ID NO:61), with portions of the tez1 sequence (SEQ IDNO:63), Est2p (SEQ ID NO:64), and the Euplotes p123 (SEQ ID NO:65).

[0071]FIG. 26 shows the DNA sequence of Est2 (SEQ ID NO:66).

[0072]FIG. 27 shows the amino acid sequence of a cDNA clone (SEQ IDNO:67) encoding human telomerase peptide motifs.

[0073]FIG. 28 shows the DNA sequence of a cDNA clone (SEQ ID NO:62)encoding human telomerase peptide motifs.

[0074]FIG. 29 shows the amino acid sequence of tez1 (SEQ ID NO:68).

[0075]FIG. 30 shows the DNA sequence of tez1 (SEQ ID NO:69).

[0076]FIG. 31 shows the alignment of EST2p (SEQ ID NO:83), Euplotes (SEQID NO:84), and Tetrahymena (SEQ ID NO:85) sequences, as well asconsensus sequence.

[0077]FIG. 32 shows the sequences of peptides useful for production ofantibodies.

[0078]FIG. 33 is a schematic summary of the tez1⁺sequencing experiments.

[0079]FIG. 34 shows two degenerate primers used in PCR to identify theS. pombe homolog of the E. aediculatus p123 sequences.

[0080]FIG. 35 shows the four major bands produced in PCR using thedegenerate primers.

[0081]FIG. 36 shows the alignment of the M2 PCR product with E.aediculatus p123, S. cerevisiae, and Oxytricha telomerase proteinsequences.

[0082]FIG. 37 is a schematic showing the 3′ RT PCR strategy.

[0083]FIG. 38 shows the libraries and the results of screening librariesfor S. pombe telomerase protein sequences.

[0084]FIG. 39 shows the results obtained with the HindIII-digestedpositive genomic clones containing S. pombe telomerase sequence.

[0085]FIG. 40 is a schematic showing the 5′ RT PCR strategy.

[0086]FIG. 41 shows the alignment of RT domains from telomerasecatalytic subunits.

[0087]FIG. 42 shows the alignment of three telomerase sequences.

[0088]FIG. 43 shows the disruption strategy used with the telomerasegenes in S. pombe.

[0089]FIG. 44 shows the experimental results confirming disruption oftez1.

[0090]FIG. 45 shows the progressive shortening of telomeres in S. pombedue to tez1 disruption.

[0091]FIG. 46 shows the DNA (SEQ ID NO:69) and amino acid (SEQ ID NO:68)sequence of tez1, with the coding regions indicated.

[0092]FIG. 47 shows the DNA (SEQ ID NO:100) and amino acid (SEQ IDNO:101) of the ORF encoding an approximately 63 kDa telomerase proteinor fragment thereof.

[0093]FIG. 48 shows an alignment of reverse transcriptase motifs fromvarious sources.

[0094]FIG. 49 provides a restriction and function map of plasmidpGRN121.

[0095]FIG. 50 provides the results of preliminary nucleic acidsequencing analysis of human telomerase (SEQ ID NO:113).

[0096]FIG. 51 provides the preliminary nucleic acid (SEQ ID NO:113) anddeduced ORF sequences (SEQ ID NOS:114-116) of human telomerase.

[0097]FIG. 52 provides a refined restriction and function map of plasmidpGRN121.

[0098]FIG. 53 provides the nucleic acid (SEQ ID NO: 117) and deduced ORFsequence (SEQ ID NO:118) of human telomerase.

[0099]FIG. 54 provides a restriction map of lambda clone 25-1.1 (ATCCaccession #______).

[0100] Definitions

[0101] To facilitate understanding the invention, a number of terms aredefined below.

[0102] As used herein, the term “ciliate” refers to any of theprotozoans belonging to the phylum Ciliaphora.

[0103] As used herein, the term “eukaryote” refers to organismsdistinguishable from “prokaryotes.” It is intended that the termencompass all organisms with cells that exhibit the usualcharacteristics of eukaryotes such as the presence of a true nucleusbounded by a nuclear membrane, within which lie the chromosomes, thepresence of membrane-bound organelles, and other characteristicscommonly observed in eukaryotic organisms. Thus, the term includes, butis not limited to such organisms as fungi, protozoa, and animals (e.g.,humans).

[0104] As used herein, the term “polyploid” refers to cells or organismswhich contain more than two sets of chromosomes.

[0105] As used herein, the term “macronucleus” refers to the larger ofthe two types of nuclei observed in the ciliates. This structure is alsosometimes referred to as the “vegetative” nucleus. Macronuclei containmany copies of each gene and are transcriptionally active.

[0106] As used herein, the term “micronucleus” refers to the smaller ofthe two types of nuclei observed in the ciliates. This structure issometimes referred to as the “reproductive” nucleus, as it participatesin meiosis and autogamy. Micronuclei are diploid and aretranscriptionally inactive.

[0107] As used herein, the term “ribonucleoprotein” refers to a complexmacromolecule containing both RNA and protein.

[0108] As used herein, the term “telomerase polypeptide,” refers to apolypeptide which is at least a portion of the Euplotes telomerasestructure. The term encompasses the 123 kDa and 43 kDa polypeptide orprotein subunits of the Euplotes telomerase. It is also intended thatthe term encompass variants of these protein subunits. It is furtherintended to encompass the polypeptides encoded by SEQ ID NOS: 1 and 3.As molecular weight measurements may vary, depending upon the techniqueused, it is not intended that the present invention be precisely limitedto the 123 kDa or 43 kDa molecular masses of the polypeptides encoded bySEQ ID NOS:1 and 3, as determined by any particular method such asSDS-PAGE.

[0109] As used herein, the terms “telomerase” and “telomerase complex”refer to functional telomerase enzymes. It is intended that the termsencompass the complex of proteins and nucleic acids found intelomerases. For example, the terms encompass the 123 kDa and 43 kDatelomerase protein subunits and RNA of E. aediculatus.

[0110] As used herein, the term “capable of replicating telomeric DNA”refers to functional telomerase enzymes which are capable of performingthe function of replicating DNA located in telomeres. It is contemplatedthat this term encompass the replication of telomeres, as well assequences and structures that are commonly found located in telomericregions of chromosomes. For example, “telomeric DNA” includes, but isnot limited to the tandem array of repeat sequences found in thetelomeres of most organisms.

[0111] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.Similarly, “amino acid sequence” as used herein refers to peptide orprotein sequence. “Peptide nucleic acid” as used herein refers to anoligomeric molecule in which nucleosides are joined by peptide, ratherthan phosphodiester, linkages. These small molecules, also designatedanti-gene agents, stop transcript elongation by binding to theircomplementary (template) strand of nucleic acid (Nielsen et al.,Anticancer Drug Des 8:53-63 [1993]).

[0112] A “deletion” is defined as a change in either nucleotide or aminoacid sequence in which one or more nucleotides or amino acid residues,respectively, are absent.

[0113] An “insertion” or “addition” is that change in a nucleotide oramino acid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to,naturally occurring sequences.

[0114] A “substitution” results from the replacement of one or morenucleotides or amino acids by different nucleotides or amino acids,respectively.

[0115] As used herein, the term “purified” refers to the removal ofcontaminant(s) from a sample. As used herein, the term “substantiallypurified” refers to molecules, either nucleic or amino acid sequences,that are removed from their natural environment, isolated or separated,and are at least 60% free, preferably 75% free, and most preferably 90%free from other components with which they are naturally associated. An“isolated polynucleotide” is therefore a substantially purifiedpolynucleotide.

[0116] As used herein, the term “probe” refers to an oligonucleotide(i.e., a sequence of nucleotides), whether occurring naturally as in apurified restriction digest or produced synthetically, which is capableof hybridizing to another oligonucleotide or polynucleotide of interest.Probes are useful in the detection, identification and isolation ofparticular gene sequences. It is contemplated that any probe used in thepresent invention will be labelled with any “reporter molecule,” so thatis detectable in any detection system, including, but not limited toenzyme (e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is furthercontemplated that the oligonucleotide of interest (i.e., to be detected)will be labelled with a reporter molecule. It is also contemplated thatboth the probe and oligonucleotide of interest will be labelled. It isnot intended that the present invention be limited to any particulardetection system or label.

[0117] As used herein, the term “target” refers to the region of nucleicacid bounded by the primers used for polymerase chain reaction. Thus,the “target” is sought to be sorted out from other nucleic acidsequences. A “segment” is defined as a region of nucleic acid within thetarget sequence. “Amplification” is defined as the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) or other technologies wellknown in the art (e.g., Dieffenbach and Dveksler, PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview N.Y. [1995]). Asused herein, the term “polymerase chain reaction” (“PCR”) refers to themethod of K. B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202, herebyincorporated by reference), which describe a method for increasing theconcentration of a segment of a target sequence in a mixture of genomicDNA without cloning or purification. This process for amplifying thetarget sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed totheir complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one “cycle”; there canbe numerous “cycles”) to obtain a high concentration of an amplifiedsegment of the desired target sequence.

[0118] The length of the amplified segment of the desired targetsequence is determined by the relative positions of the primers withrespect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified”.

[0119] As used herein, the term “polymerase” refers to any polymerasesuitable for use in the amplification of nucleic acids of interest. Itis intended that the term encompass such DNA polymerases as Taq DNApolymerase obtained from Thermus aquaticus, although other polymerases,both thermostable and thermolabile are also encompassed by thisdefinition.

[0120] With PCR, it is possible to amplify a single copy of a specifictarget sequence in genomic DNA to a level detectable by severaldifferent methodologies (e.g., hybridization with a labeled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide sequence can be amplifiedwith the appropriate set of primer molecules. In particular, theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications. Amplified targetsequences may be used to obtain segments of DNA (e.g., genes) forinsertion into recombinant vectors.

[0121] As used herein, the terms “PCR product” and “amplificationproduct” refer to the resultant mixture of compounds after two or morecycles of the PCR steps of denaturation, annealing and extension arecomplete. These terms encompass the case where there has beenamplification of one or more segments of one or more target sequences.

[0122] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to bacterial enzymes, each of which cutdouble-stranded DNA at or near a specific nucleotide sequence.

[0123] As used herein, the term “recombinant DNA molecule” as usedherein refers to a DNA molecule which is comprised of segments of DNAjoined together by means of molecular biological techniques.

[0124] As used herein, the terms “complementary” or “complementarity”are used in reference to polynucleotides (i.e., a sequence ofnucleotides) related by the base-pairing rules. For example, for thesequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods which depend uponbinding between nucleic acids.

[0125] The term “homology” refers to a degree of complementarity. Theremay be partial homology or complete homology (i.e., identity). Apartially complementary sequence is one that at least partially inhibitsa completely complementary sequence from hybridizing to a target nucleicacid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target whichlacks even a partial degree of complementarity (e.g., less than about30% identity); in the absence of non-specific binding the probe will nothybridize to the second non-complementary target.

[0126] The art knows well that numerous equivalent conditions may beemployed to comprise either low or high stringency conditions; factorssuch as the length and nature (DNA, RNA, base composition) of the probeand nature of the target (DNA, RNA, base composition, present insolution or immobilized, etc.) and the concentration of the salts andother components (e.g., the presence or absence of formamide, dextransulfate, polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency hybridization different from, but equivalent to, the abovelisted conditions. The term “hybridization” as used herein includes “anyprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs, Dictionary of Biotechnology,Stockton Press, New York N.Y. [1994].

[0127] “Stringency” typically occurs in a range from about T_(m)−5° C.(5° C. below the Tm of the probe) to about 20° C. to 25° C. below T_(m).As will be understood by those of skill in the art, a stringenthybridization can be used to identify or detect identical polynucleotidesequences or to identify or detect similar or related polynucleotidesequences.

[0128] As used herein, the term “T_(m)” is used in reference to the“melting temperature.” The melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf dissociated into single strands. The equation for calculating theTm of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the Tm value may be calculated by theequation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridisation, in Nucleic Acid Hybridisation (1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m).

[0129] As used herein the term “hybridization complex” refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bounds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleic,acid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C_(o)t or R_(o)tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized to a solid support (e.g., anylon membrane or a nitrocellulose filter as employed in Southern andNorthern blotting, dot blotting or a glass slide as employed in in situhybridization, including FISH [fluorescent in situ hybridization]).

[0130] As used herein, the term “antisense” is used in reference to RNAsequences which are complementary to a specific RNA sequence (e.g.,mRNA). Antisense RNA may be produced by any method, including synthesisby splicing the gene(s) of interest in a reverse orientation to a viralpromoter which permits the synthesis of a coding strand. Once introducedinto a cell, this transcribed strand combines with natural mRNA producedby the cell to form duplexes. These duplexes then block either thefurther transcription of the mRNA or its translation. In this manner,mutant phenotypes may be generated. The term “antisense strand” is usedin reference to a nucleic acid strand that is complementary to the“sense's strand. The designation (-) (i.e., “negative”) is sometimesused in reference to the antisense strand, with the designation (+)sometimes used in reference to the sense (i.e., “positive”) strand.

[0131] As used herein the term “portion” when in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:2” encompasses the full-length 123 kDa telomerase protein subunit andfragments thereof.

[0132] The term “antigenic determinant” as used herein refers to thatportion of an antigen that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0133] The terms “specific binding” or specifically binding” when usedin reference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labelled “A” and the antibody will reduce the amount oflabelled A bound to the antibody.

[0134] The term “sample” as used herein is used in its broadest sense. Abiological sample suspected of containing nucleic acid encodingtelomerase subunits may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern blot analysis), RNA (insolution or bound to a solid support such as for Northern blotanalysis), cDNA (in solution or bound to a solid support) and the like.A sample suspected of containing a protein may comprise a cell, aportion of a tissue, an extract containing one or more proteins and thelike.

[0135] The term “correlates with expression of a polynucleotide,” asused herein, indicates that the detection of the presence of ribonucleicacid (RNA) complementary to a telomerase sequence by hybridizationassays is indicative of the presence of mRNA encoding eukaryotictelomerases, including human telomerases in a sample, and therebycorrelates with expression of the telomerase mRNA from the gene encodingthe protein.

[0136] “Alterations in the polynucleotide” as used herein comprise anyalteration in the sequence of polynucleotides encoding telomerases,including deletions, insertions, and point mutations that may bedetected using hybridization assays. Included within this definition isthe detection of alterations to the genomic DNA sequence which encodestelomerase (e.g., by alterations in pattern of restriction enzymefragments capable of hybridizing to any sequence such as SEQ ID NOS: 1or 3 [e.g., RFLP analysis], the inability of a selected fragment of anysequence to hybridize to a sample of genomic DNA [e.g., usingallele-specific oligonucleotide probes], improper or unexpectedhybridization, such as hybridization to a locus other than the normalchromosomal locus for the telomere or telomerase genes e.g., using FISHto metaphase chromosomes spreads, etc.]).

[0137] A “variant” in regard to amino acid sequences is used to indicatean amino acid sequence that differs by one or more amino acids fromanother, usually related amino acid. The variant may have “conservative”changes, wherein a substituted amino acid has similar structural orchemical properties (e.g., replacement of leucine with isoleucine). Morerarely, a variant may have “non-conservative” changes, e.g., replacementof a glycine with a tryptophan. Similar minor variations may alsoinclude amino acid deletions or insertions (i.e., additions), or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNAStar software. Thus, it is contemplated thatthis definition will encompass variants of telomerase and/or telomeraseprotein subunits. For example, the polypeptides encoded by the threeopen reading frames (ORFs) of the 43 kDa polypeptide gene may beconsidered to be variants of each other. Such variants can be tested infunctional assays, such as telomerase assays to detect the presence offunctional telomerase in a sample.

[0138] The term “derivative” as used herein refers to the chemicalmodification of a nucleic acid encoding telomerase structures, such asthe 123 kDa or 43 kDa protein subunits of the E. aediculatus telomerase,or other telomerase proteins or peptides. Illustrative of suchmodifications would be replacement of hydrogen by an alkyl, acyl, oramino group. A nucleic acid derivative would encode a polypeptide whichretains essential biological characteristics of naturally-occurringtelomerase or its subunits.

[0139] The term “biologically active” refers to telomerase molecules orpeptides having structural, regulatory, or biochemical functions of anaturally occurring telomerase molecules or peptides. Likewise,“immunologically active,” defines the capability of the natural,recombinant, or synthetic telomerase proteins or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells, and to bind with specific antibodies.

[0140] “Affinity purification” as used herein refers to the purificationof ribonucleoprotein particles, through the use of an “affinityoligonucleotide” (i.e., an antisense oligonucleotides) to bind theparticle, followed by the step of eluting the particle from theoligonucleotide by means of a “displacement oligonucleotide.” In thepresent invention, the displacement oligonucleotide has a greater degreeof complementarity with the affinity oligonucleotide, and thereforeproduces a more thermodynamically stable duplex than the particle andthe affinity oligonucleotide. For example, telomerase may be bound tothe affinity oligonucleotide and then eluted by use of a displacementoligonucleotide which binds to the affinity oligonucleotide. In essence,the displacement oligonucleotide displaces the telomerase from theaffinity oligonucleotide, allowing the elution of the telomerase. Undersufficiently mild conditions, the method results in the enrichment offunctional ribonucleoprotein particles. Thus, the method is useful forthe purification of telomerase from a mixture of compounds.

GENERAL DESCRIPTION OF THE INVENTION

[0141] The present invention provides purified telomerase preparationsand telomerase protein subunits useful for investigations of theactivities of telomerases, including potential nuclease activities. Inparticular, the present invention is directed to the telomerase andco-purifying polypeptides obtained from Euplotes aediculatus. Thisorganism, a hypotrichous ciliate, was chosen for use in this inventionas it contains an unusually large number of chromosomal ends (Prescott,Microbiol. Rev., 58:233 [1994]), because a very large number ofgene-sized DNA molecules are present in its polyploid macronucleus.Tetrahymena, a holotrichous ciliate commonly used in previous studies oftelomerase and telomeres, is as evolutionarily distant from Euplotes asplants are from mammals (Greenwood et al., J. Mol. Evol., 3:163 [1991]).

[0142] The homology found between the 123 kDa E. aediculatus telomerasesubunit and the L8543.12 sequence (i.e., Est2 of Saccharomycescerevisiae; See, Lendvay et al., Genetics 144:1399-1412 [1996]),Schizosaccharomyces, and human motifs, provides a strong basis forpredicting that full human telomerase molecule comprises a protein thatis large, basic, and includes such reverse transcriptase motifs. Thus,the compositions and methods of the present invention is useful for theidentification of other telomerases, from a wide variety of species. Thepresent invention describes the use of the 123 kDa reverse transcriptasemotifs in a method to identify similar motifs in organisms that aredistantly related to Euplotes (e.g., Oxytricha), as well as organismsthat are not related to Euplotes (e.g., Saccharomyces,Schizosaccharomyces, humans, etc.).

[0143] The present invention also provides additional methods for thestudy of the structure and function of distinct forms of telomerase. Itis contemplated that the telomerase proteins of the present inventionwill be useful in diagnostic applications, evolutionary (e.g.,phylogenetic) investigations, as well as development of compositions andmethods for cancer therapy or anti-aging regimens. Although thetelomerase protein subunits of the present invention themselves haveutility, it further contemplated that the polypeptides of the presentinvention will be useful in conjunction with the RNA moiety of thetelomerase enzyme (i.e., a complete telomerase).

[0144] It is also contemplated that methods and compositions of thisinvention will lead to the discovery of additional unique telomerasestructures and/or functions. In addition, the present invention providesnovel methods for purification of functional telomerase, as well astelomerase proteins. This affinity based method described in Example 3,is an important aspect in the purification of functionally activetelomerase. A key advantage of this procedure is the ability to use mildelution conditions, during which proteins that bind non-specifically tothe column matrix are not eluted.

DETAILED DESCRIPTION OF THE INVENTION

[0145] The present invention is directed to the nucleic and amino acidsequences of the protein subunits of the E. aediculatus telomerase, aswell as the nucleic and amino acid sequences of the telomerases fromother organisms, including humans. In addition, the present invention isdirected to the purification of functional telomerase. As describedbelow the present invention also comprises various forms of telomerase,including recombinant telomerase and telomerase protein subunits,obtained from various organisms.

[0146] The 123 kDa and 43 kDa Telomerase Subunit Protein Sequences

[0147] The nucleic acid and deduced amino acid sequences of the 123 and43 kDa protein subunits are shown in FIGS. 1-6. In accordance with theinvention, any nucleic acid sequence which encodes E. aediculatustelomerase or its subunits can be used to generate recombinant moleculeswhich express the telomerase or its subunits.

[0148] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of telomerasesubunit protein sequences, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. The invention contemplates each and every possible variationof nucleotide sequence that could be made by selecting combinationsbased on possible codon choices, taking into account the use of thecodon “UGA” as encoding cysteine in E. aediculatus . Other than theexception of the “UGA” codon, these combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence encoding naturally occurring E. aediculatus telomerase, and allsuch variations are to be considered as being specifically disclosed.For example, the amino acid sequences encoded by each of the three openreading frames of the 43 kDa nucleotide sequence are specificallyincluded (SEQ ID NOS:4-6). It is contemplated that any variant forms oftelomerase subunit protein be encompassed by the present invention, aslong as the proteins are functional in assays such as those described inthe Examples.

[0149] Although nucleotide sequences which encode E. aediculatustelomerase protein subunits and their variants are preferably capable ofhybridizing to the nucleotide sequence of the naturally occurringsequence under appropriately selected conditions of stringency, it maybe advantageous to produce nucleotide sequences encoding E. aediculatustelomerase protein subunits or their derivatives possessing asubstantially different codon usage, including the “standard” codonusage employed by human and other systems. Codons may be selected toincrease the rate at which expression of the peptide occurs in aparticular prokaryotic or eukaryotic expression host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding telomerase subunits and their derivatives without altering theencoded amino acid sequences include the production of RNA transcriptshaving more desirable properties, such as a greater or a shorterhalf-life, than transcripts produced from the naturally occurringsequence.

[0150] It is now possible to produce a DNA sequence, or portionsthereof, encoding telomerase protein subunits and their derivativesentirely by synthetic chemistry, after which the synthetic gene may beinserted into any of the many available DNA vectors and cell systemsusing reagents that are well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encoding E.aediculatus protein subunits or any portion thereof, as well assequences encoding yeast or human telomerase proteins, subunits, or anyportion thereof.

[0151] Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequence of FIGS. 9, 11, 12, and 26, under various conditionsof stringency. Hybridization conditions are based on the meltingtemperature (T_(m)) of the nucleic acid binding complex or probe, astaught in Berger and Kimmel (Berger and Kimmel, Guide to MolecularCloning Techniques, Meth. Enzymol., vol. 152, Academic Press, San DiegoCalif. [1987]) incorporated herein by reference, and may be used at adefined “stringency”.

[0152] Altered nucleic acid sequences encoding telomerase proteinsubunits which may be used in accordance with the invention includedeletions, insertions or substitutions of different nucleotidesresulting in a polynucleotide that encodes the same or a functionallyequivalent telomerase subunit. The protein may also show deletions,insertions or substitutions of amino acid residues which produce asilent change and result in a functionally equivalent telomerasesubunit. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of the telomerase subunit is retained. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine;glycine, alanine; asparagine, glutamine; serine, threonine; andphenylalanine, tyrosine.

[0153] Methods for DNA sequencing are well known in the art and employsuch enzymes as the Klenow fragment of DNA polymerase I, Sequenase® (USBiochemical Corp, Cleveland Ohio), Taq DNA polymerase (Perkin Elmer,Norwalk Conn.), thermostable T7 polymerase (Amersham, Chicago Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

[0154] Also included within the scope of the present invention arealleles encoding human telomerase proteins and subunits. As used herein,the term “allele” or “allelic sequence” is an alternative form of thenucleic acid sequence encoding human telomerase proteins or subunits.Alleles result from mutations (i.e., changes in the nucleic acidsequence), and generally produce altered mRNAs or polypeptides whosestructure and/or function may or may not be altered. An given gene mayhave no, one or many allelic forms. Common mutational changes that giverise to alleles are generally ascribed to natural deletions, additions,or substitutions of amino acids. Each of these types of changes mayoccur alone, or in combination with the others, one or more times withina given sequence.

[0155] Human Telomerase Motifs

[0156] The present invention also provides nucleic and amino acidsequence information for human telomerase motifs. These sequences werefirst identified in a BLAST search conducted using the Euplotes 123 kDapeptide, and a homologous sequence from Schizosaccharomyces, designatedas “tez1.” FIG. 25 shows the sequence alignment of the Euplotes(“p123”), Schizosaccharomyces (“tezl”), Est2p (i.e., the S. cerevisiaeprotein encoded by the Est2 nucleic acid sequence, and also referred toherein as “L8543.12”), and the human homolog identified in thiscomparison search. The amino acid sequence of this aligned portion isprovided in SEQ ID NO:61 (the cDNA sequence is provided in SEQ IDNO:62), while the portion of tez1 shown in FIG. 25 is provided in SEQ IDNO:63. The portion of Est2 shown in this Figure is also provided in SEQID NO:64, while the portion of p123 shown is also provided in SEQ IDNO:65.

[0157] As shown in FIG. 25, there are regions that are highly conservedamong these proteins. For example, as shown in this Figure, there areregions of identity in “Motif 0,” “Motif 1, “Motif 2,” and “Motif 3.”The identical amino acids are indicated with an asterisk (*), while thesimilar amino acid residues are indicated by a circle (). Thisindicates that there are regions within the telomerase motifs that areconserved among a wide variety of eukaryotes, ranging from yeast tociliates, to humans. It is contemplated that additional organisms willlikewise contain such conserved regions of sequence.

[0158]FIG. 27 shows the amino acid sequence of the cDNA clone encodinghuman telomerase motifs (SEQ ID NO:67), while FIG. 28 shows the DNAsequence of the clone. FIG. 29 shows the amino acid sequence of tez1(SEQ ID NO:68), while FIG. 30 shows the DNA sequence of tez1 (SEQ IDNO:69). In FIG. 30, the introns and other non-coding regions are shownin lower case, while the exons (i.e., coding regions are shown in uppercase

[0159] Extending The Polynucleotide Sequence

[0160] The polynucleotide sequence encoding telomerase, or telomeraseprotein subunits, or their functional equivalents, may be extendedutilizing partial nucleotide sequence and various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, Gobinda et al. (Gobinda et al., PCR Meth. Applic.2:318-22 [1993]) describe “restriction-site” polymerase chain reaction(PCR) as a direct method which uses universal primers to retrieveunknown sequence adjacent to a known locus. First, genomic DNA isamplified in the presence of primer to a linker sequence and a primerspecific to the known region. The amplified sequences are subjected to asecond round of PCR with the same linker primer and another specificprimer internal to the first one. Products of each round of PCR aretranscribed with an appropriate RNA polymerase and sequenced usingreverse transcriptase.

[0161] Inverse PCR can be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia et al., Nucleic AcidsRes 16:8186 [1988]). The primers may be designed using OLIGO® 4.06Primer Analysis Software (National Biosciences Inc, Plymouth Minn.[1992]), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0162] Capture PCR (Lagerstrom et al. PCR Methods Applic 1:111-19[1991]), a method for PCR amplification of DNA fragments adjacent to aknown sequence in human and yeast artificial chromosome DNA, may also beused. Capture PCR also requires multiple restriction enzyme digestionsand ligations to place an engineered double-stranded sequence into anunknown portion of the DNA molecule before PCR.

[0163] Another method which may be used to retrieve unknown sequence iswalking PCR (Parker et al., Nucleic Acids Res 19:3055-60 [1991]), amethod for targeted gene walking. Alternatively, PCR, nested primers,PromoterFinder™ (Clontech, Palo Alto Calif.) and PromoterFinderlibraries can be used to walk in genomic DNA. This process avoids theneed to screen libraries and is useful in finding intron/exon junctions.

[0164] Preferred libraries for screening for full length cDNAs are onesthat have been size-selected to include larger cDNAs. Also, randomprimed libraries are preferred in that they will contain more sequenceswhich contain the 5′ and upstream regions of genes. A randomly primedlibrary may be particularly useful if an oligo d(T) library does notyield a full-length cDNA. Genomic libraries are useful for extensioninto the 5′ nontranslated regulatory region.

[0165] Capillary electrophoresis may be used to analyze either the sizeor confirm the nucleotide sequence in sequencing or PCR products.Systems for rapid sequencing are available from Perkin Elmer, BeckmanInstruments (Fullerton Calif.), and other companies. Capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity is converted to electricalsignal using appropriate software (e.g., Genotyper™ and SequenceNavigator™ from Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display is computercontrolled. Capillary electrophoresis is particularly suited to thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample. The reproducible sequencing of up to 350bp of M13 phage DNA in 30 min has been reported (Ruiz-Martinez et al.,Anal Chem 65:2851-8 [1993]).

[0166] Expression of the Nucleotide Sequence

[0167] In accordance with the present invention, polynucleotidesequences which encode telomerase, telomerase protein subunits, or theirfunctional equivalents, may be used in recombinant DNA molecules thatdirect the expression of telomerase or telomerase subunits byappropriate host cells.

[0168] The nucleotide sequences of the present invention can beengineered in order to alter either or both telomerase subunits for avariety of reasons, including but not limited to, alterations whichmodify the cloning, processing and/or expression of the gene product.For example, mutations may be introduced using techniques which are wellknown in the art (e.g., site-directed mutagenesis to insert newrestriction sites, to alter glycosylation patterns, to change codonpreference, to produce splice variants, etc.).

[0169] In an alternate embodiment of the invention, the sequenceencoding the telomerase subunit(s) may be synthesized, whole or in part,using chemical methods well known in the art (See e.g., Caruthers etal., Nucleic Acids Res. Symp. Ser., 215-223 [1980]; and Horn et al.Nucleic Acids Res. Symp. Ser., 225-232 [1980]). Alternatively, theprotein itself could be produced using chemical methods to synthesize atelomerase subunit amino acid sequence, in whole or in part. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, et al. Science 269:202 [1995]) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

[0170] The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton,Proteins, Structures and Molecular Principles, WH Freeman and Co, NewYork N.Y. [1983]). The composition of the synthetic peptides may beconformed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally the amino acidsequences of telomerase subunit proteins, or any part thereof, may bealtered during direct synthesis and/or combined using chemical methodswith sequences from other proteins, or any part thereof, to produce avariant polypeptide.

[0171] Expression Systems

[0172] In order to express a biologically active telomerase proteinsubunit, the nucleotide sequence encoding the subunit or the functionalequivalent, is inserted into an appropriate expression vector (i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence). In order to express abiologically active telomerase enzyme, the nucleotide sequence encodingthe telomerase protein subunits are inserted into appropriate expressionvectors and the nucleotide sequence encoding the telomerase RNA subunitis inserted into the same or another vector for RNA expression. Theprotein and RNA subunits are then either expressed in the same cell orexpressed separately, and then mixed to achieve a reconstitutedtelomerase.

[0173] Methods which are well known to those skilled in the art can beused to construct expression vectors containing a telomerase proteinsubunit sequence and appropriate transcriptional or translationalcontrols. These methods include in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination or genetic recombination.Such techniques are described in Sambrook et al. (Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview NY [1989]), and Ausubel et al. (Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York N.Y.[1989]). These same methods may be used to convert the UGA codons, whichencode cysteine in Euplotes, to the UGU or UGC codon for cysteinerecognized by the host expression system.

[0174] A variety of expression vector/host systems may be utilized tocontain and express a telomerase subunit-encoding sequence. Theseinclude but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti orpBR322 plasmid); or animal cell systems.

[0175] The “control elements” or “regulatory sequences” of these systemsvary in their strength and specificities and are those non-translatedregions of the vector, enhancers, promoters, and 3′ untranslatedregions, which interact with host cellular proteins to carry outtranscription and translation. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla Calif.) or pSportl (Gibco BRL) and ptrp-lac hybrids and the likemay be used. The baculovirus polyhedrin promoter may be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO; and storage protein genes) or from plantviruses (e.g., viral promoters or leader sequences) may be cloned intothe vector. In mammalian cell systems, promoters from the mammaliangenes or from mammalian viruses are most appropriate. If it is necessaryto generate a cell line that contains multiple copies of the sequenceencoding telomerase or telomerase protein subunits, vectors based onSV40 or EBV may be used with an appropriate selectable marker.

[0176] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for the telomerase protein orsubunit. For example, when large quantities of telomerase protein,subunit, or peptides, are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be desirable. Such vectors include, but are notlimited to, the multifunctional E. coli cloning and expression vectorssuch as Bluescript® (Stratagene), in which the sequence encoding thetelomerase or protein subunit may be ligated into the vector in framewith sequences for the amino-terminal Met and the subsequent 7 residuesof β-galactosidase so that a hybrid protein is produced (e.g., pINvectors; Van Heeke and Schuster, J. Biol. Chem., 264:5503-5509 [1989])and the like. pGEX vectors (Promega, Madison Wis.) may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems are designed to includeheparin, thrombin or factor Xa protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

[0177] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al., Meth. Enzymol., 153:516-544 (1987).

[0178] In cases where plant expression vectors are used, the expressionof a sequence encoding telomerase or protein subunit, may be driven byany of a number of promoters. For example, viral promoters such as the35S and 19S promoters of CaMV (Brisson et al., Nature 310:511-514[1984]) may be used alone or in combination with the omega leadersequence from TMV (Takamatsu et al., EMBO J., 6:307-311 [1987]).Alternatively, plant promoters such as the small subunit of RUBISCO(Coruzzi et al. EMBO J., 3:1671-1680 [1984]; Broglie et al., Science224:838-843 [1984]) or heat shock promoters (Winter and SinibaldiResults Probl. Cell Differ., 17:85-105 [1991]) may be used. Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection (for reviews of suchtechniques, see Hobbs or Murry, in McGraw Hill Yearbook of Science andTechnology McGraw Hill New York N.Y., pp. 191-196 [1992]; or Weissbachand Weissbach, Methods for Plant Molecular Biology, Academic Press, NewYork N.Y., pp. 421-463 [1988]).

[0179] An alternative expression system which could be used to expresstelomerase or telomerase protein subunit is an insect system. In onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequence encoding the telomerasesequence of interest may be cloned into a nonessential region of thevirus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the sequence encoding thetelomerase protein or telomerase protein subunit will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses are then used to infect S. frugiperdacells or Trichoplusia larvae in which the telomerase sequence isexpressed (Smith et al., J. Virol., 46:584 [1983]; Engelhard et al.,Proc. Natl. Acad. Sci. 91:3224-7 [1994]).

[0180] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, a sequence encoding telomerase protein or telomeraseprotein subunit, may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a nonessential El or E3 regionof the viral genome will result in a viable virus capable of expressingin infected host cells (Logan and Shenk, Proc. Natl. Acad. Sci.,81:3655-59 [1984]). In addition, transcription enhancers, such as theRous sarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

[0181] Specific initiation signals may also be required for efficienttranslation of a sequence encoding telomerase protein subunits. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where the sequence encoding a telomerase protein subunit, itsinitiation codon and upstream sequences are inserted into the mostappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only coding sequence, ora portion thereof, is inserted, exogenous transcriptional controlsignals including the ATG initiation codon must be provided.Furthermore, the initiation codon must be in the correct reading frameto ensure transcription of the entire insert. Exogenous transcriptionalelements and initiation codons can be of various origins, both naturaland synthetic. The efficiency of expression may be enhanced by theinclusion of enhancers appropriate to the cell system in use (Scharf etal., Results Probl. Cell Differ., 20:125 [1994]; and Bittner et al.,Meth. Enzymol., 153:516 [1987).

[0182] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO (ATCC CCL 61 and CRL 9618),HeLa (ATCC CCL 2), MDCK (ATCC CCL 34 and CRL 6253), HEK 293 (ATCC CRL1573), WI-38 (ATCC CCL 75) (ATCC: American Type Culture Collection,Rockville, Md.), etc have specific cellular machinery and characteristicmechanisms for such post-translational activities and may be chosen toensure the correct modification and processing of the introduced,foreign protein.

[0183] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress telomerase or a telomerase subunit protein may be transformedusing expression vectors which contain viral origins of replication orendogenous expression elements and a selectable marker gene. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clumps ofstably transformed cells can be proliferated using tissue culturetechniques appropriate to the cell type.

[0184] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-32[1977]) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817[1980]) genes which can be employed in tk- or aprt- cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci., 77:3567 [1980]);npt, which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin et al., J. Mol. Biol., 150:1 [1981]) and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, In McGraw Hill Yearbook ofScience and Technology, McGraw Hill, New York N.Y., pp 191-196, [1992]).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartmanand Mulligan, Proc. Natl. Acad. Sci., 85:8047 [1988]). Recently, the useof visible markers has gained popularity with such markers asanthocyanins, β glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes etal., Meth. Mol. Biol., 55:121 [1995]).

[0185] Identification of Transformants Containing the PolynucleotideSequence

[0186] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionshould be confirmed. For example, if the sequence encoding a telomeraseprotein subunit is inserted within a marker gene sequence, recombinantcells containing the sequence encoding the telomerase protein subunitcan be identified by the absence of marker gene function. Alternatively,a marker gene can be placed in tandem with the sequence encodingtelomerase protein subunit under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem sequence as well.

[0187] Alternatively, host cells which contain the coding sequence fortelomerase or a telomerase protein subunit and express the telomerase orprotein subunit be identified by a variety of procedures known to thoseof skill in the art. These procedures include, but are not limited to,DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassaytechniques which include membrane, solution, or chip-based technologiesfor the detection and/or quantification of the nucleic acid or protein.

[0188] The presence of the polynucleotide sequence encoding telomeraseprotein subunits can be detected by DNA-DNA or DNA-RNA hybridization oramplification using probes, portions, or fragments of the sequenceencoding the subunit. Nucleic acid amplification based assays involvethe use of oligonucleotides or oligomers based on the nucleic acidsequence to detect transformants containing DNA or RNA encoding thetelomerase subunit. As used herein “oligonucleotides” or “oligomers”refer to a nucleic acid sequence of approximately 10 nucleotides orgreater and as many as approximately 100 nucleotides, preferably between15 to 30 nucleotides, and more preferably between 20-25 nucleotideswhich can be used as a probe or amplimer.

[0189] A variety of protocols for detecting and measuring the expressionof proteins (e.g., telomerase or a telomerase protein subunits) usingeither polyclonal or monoclonal antibodies specific for the protein areknown in the art. Examples include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting(FACS). These and other assays are described, among other places, inHampton et al., Serological Methods a Laboratory Manual, APS Press, StPaul MN [1990]) and Maddox et al., J. Exp. Med., 158:1211 [1983]).

[0190] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting related sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, a telomerase protein subunit sequence, or anyportion of it, may be cloned into a vector for the production of an“mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3 or SP6 and labelednucleotides.

[0191] A number of companies such as Pharmacia Biotech (PiscatawayN.J.), Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio)supply commercial kits and protocols for these procedures. Suitablereporter molecules or labels include those radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241, herein incorporated by reference. Also, recombinantimmunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567incorporated herein by reference.

[0192] Purification of Recombinant Telomerase and Telomerase SubunitProteins

[0193] In addition to the method of purification described in Example 3below, it is contemplated that additional methods of purifyingrecombinantly produced telomerase or telomerase protein subunits will beused. For example, host cells transformed with a nucleotide sequenceencoding telomerase or telomerase subunit protein(s) may be culturedunder conditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein produced by a recombinant cellmay be secreted or contained intracellularly depending on the sequenceand/or the vector used. As will be understood by those of skill in theart, expression vectors containing the telomerase or subunit proteinencoding sequence can be designed with signal sequences which directsecretion of the telomerase or telomerase subunit protein through aprokaryotic or eukaryotic cell membrane. Other recombinant constructionsmay join the sequence encoding the telomerase or subunit protein to anucleotide sequence encoding a polypeptide domain.

[0194] Telomerase or telomerase subunit protein(s) may also be expressedas recombinant proteins with one or more additional polypeptide domainsadded to facilitate protein purification. Such purification facilitatingdomains include, but are not limited to, metal chelating peptides suchas polyhistidine tracts and histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequences such as Factor Xaor enterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and telomerase or telomerase protein subunits is useful tofacilitate purification. One such expression vector provides forexpression of a fusion protein comprising the sequence encodingtelomerase or telomerase protein subunits and nucleic acid sequenceencoding 6 histidine residues followed by thioredoxin and anenterokinase cleavage site. The histidine residues facilitatepurification while the enterokinase cleavage site provides a means forpurifying the telomerase or telomerase protein subunit from the fusionprotein. Literature pertaining to vectors containing fusion proteins isavailable in the art (See e.g., Kroll et al., DNA Cell. Biol., 12:441-53[1993]).

[0195] In addition to recombinant production, fragments of telomerasesubunit protein may be produced by direct peptide synthesis usingsolid-phase techniques (See e.g., Merrifield, J. Am. Chem. Soc., 85:2149[1963]). In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431 A Peptide Synthesizer (PerkinElmer, Foster City Calif.) in accordance with the instructions providedby the manufacturer. Various fragments of telomere protein subunit maybe chemically synthesized separately and combined using chemical methodsto produce the full length molecule.

[0196] Uses of Telomerase and Telomerase Subunit Proteins

[0197] The rationale for use of the nucleotide and peptide sequencesdisclosed herein is based in part on the homology between the E.aediculatus telomerase 123 kDa protein subunit, the yeast proteinL8543.12 (Est2), Schizosaccharomyces, and the human motifs observedduring the development of the present invention. In particular, theyeast and 123 kDa protein contain the reverse transcriptase motif intheir C-terminal regions, they share similarity in regions outside thereverse transcriptase motif, they are similarly basic (with a pI of 10.1for the 123 kDa protein, and of 10.0 for the yeast), and they are bothlarge (123 kDa and 103 kDa). Furthermore, in view of the reversetranscriptase motifs, these subunits are believed to comprise thecatalytic core of their respective telomerases. Indeed, the reversetranscriptase motifs of the 123 kDa E. aediculatus telomerase proteinsubunit is shown in the present invention to be useful for theidentification of similar sequences in other organisms.

[0198] As E. aediculatus and S. cerevisiae are so phylogeneticallydistant, it is contemplated that this homology provides a strong basisfor predicting that human and other telomerases will contain a proteinthat is large, basic, and includes such reverse transcriptase motifs.Indeed, motifs have been identified within a clone encoding the humanhomolog of the telomerase protein. It is further contemplated that thisprotein is essential for human telomerase catalytic activity. Thisobservation should prove valuable for amplification of the humantelomerase gene by PCR or other methods, for screening for telomerasesequences in human and other animals, as well as for prioritizingcandidate telomerase proteins or genes identified by genetic,biochemical, or nucleic acid hybridization methods. It is alsocontemplated that the telomerase proteins of the present invention willfind use in tailing DNA 3′ ends in vitro.

[0199] It is contemplated that expression of telomerase and/ortelomerase subunit proteins in cell lines will find use in thedevelopment of diagnostics for tumors and aging factors. The nucleotidesequence may be used in hybridization or PCR technologies to diagnosethe induced expression of messenger RNA sequences early in the diseaseprocess. Likewise the protein can be used to produce antibodies usefulin ELISA assays or a derivative diagnostic format. Such diagnostic testsmay allow different classes of human tumors or other cell-proliferativediseases to be distinguished and thereby facilitate the selection ofappropriate treatment regimens.

[0200] It is contemplated that the finding of the reverse transcriptasemotifs in the telomerase proteins of the present invention will be usedto develop methods to test known and yet to be described reversetranscriptase inhibitors, including nucleosides, and non-nucleosides foranti-telomerase activity.

[0201] It is contemplated that the amino acid sequence motifs disclosedherein will lead to the development of drugs (e.g., telomeraseinhibitors) useful in humans and/or other animals, that will arrest celldivision in cancers or other disorders characterized by proliferation ofcells. It is also contemplated that the telomerase proteins will finduse in methods for targeting and directing RNA or RNA-tethered drugs tospecific sub-cellular compartments such as the nucleus or sub-nuclearorganelles, or to telomeres.

[0202] In one embodiment of the diagnostic method of the presentinvention, normal or standard values for telomerase mRNA expression areestablished as a baseline. This can be accomplished by a number ofassays such as quantitating the amount of telomerase mRNA in tissuestaken from normal subjects, either animal or human, with nucleic probesderived from the telomerase or telomerase protein subunit sequencesprovided herein (either DNA or RNA forms) using techniques which arewell known in the art (e.g., Southern blots, Northern blots, dot or slotblots). The standard values obtained from normal samples may be comparedwith values obtained from samples from subjects potentially affected bydisease (e.g., tumors or disorders related to aging). Deviation betweenstandard and subject values can establish the presence of a diseasestate. In addition, the deviation can indicate, within a disease state,a particular clinical outcome (e.g., metastatic or non-metastatic).

[0203] The nucleotide sequence encoding telomerase or telomerase proteinsubunits is useful when placed in an expression vector for makingquantities of protein for therapeutic use. The antisense nucleotidesequence of the telomerase gene is potentially useful in vectorsdesigned for gene therapy directed at neoplasia including metastases.Additionally, the inhibition of telomerase expression may be useful indetecting the development of disturbances in the aging process orproblems occurring during chemotherapy. Alternatively, the telomerase ortelomerase protein subunit encoding nucleotide sequences may used todirect the expression of telomerase or subunits in situations where itis desirable to increase the amount of telomerase activity.

[0204] Telomere Subunit Protein Antibodies

[0205] It is contemplated that antibodies directed against thetelomerase subunit proteins will find use in the diagnosis and treatmentof conditions and diseases associated with expression of telomerase(including the over-expression and the absence of expression). Suchantibodies include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fab fragments and fragments produced by a Fabexpression library. Given the phylogenetic conservation of the reversetranscriptase motif in the 123 kDa subunit of the Euplotes telomerase,it is contemplated that antibodies directed against this subunit may beuseful for the identification of homologous subunits in other organisms,including humans. It is further contemplated that antibodies directedagainst the motifs provided in the present invention will find use intreatment and/or diagnostic areas.

[0206] Telomerase subunit proteins used for antibody induction need notretain biological activity; however, the protein fragment, oroligopeptide must be immunogenic, and preferably antigenic. Peptidesused to induce specific antibodies may have an amino acid sequenceconsisting of at least five amino acids, preferably at least 10 aminoacids. Preferably, they should mimic a portion of the amino acidsequence of the natural protein and may contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches oftelomerase subunit protein amino acids may be fused with those ofanother protein such as keyhole limpet hemocyanin and antibody producedagainst the chimeric molecule. Complete telomerase used for antibodyinduction can be produced by co-expression of protein and RNA componentsin cells, or by reconstitution in vitro from components separatelyexpressed or synthesized.

[0207] For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc may be immunized by injection with telomeraseprotein, protein subunit, or any portion, fragment or oligopeptide whichretains immunogenic properties. Depending on the host species, variousadjuvants may be used to increase immunological response. Such adjuvantsare commercially available, and include but are not limited to Freund's,mineral gels such as aluminum hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (BacillusCalmette-Guerin) and Corynebacterium parvum are potentially usefuladjuvants.

[0208] Monoclonal antibodies to telomerase or telomerase proteinsubunits be prepared using any technique which provides for theproduction of antibody molecules by continuous cell lines in culture.These include but are not limited to the hybridoma technique originallydescribed by Koehler and Milstein (Koehler and Milstein, Nature256:495-497 [1975]), the human B-cell hybridoma technique (Kosbor etal., Immunol. Today 4:72 [1983]; Cote et al., Proc. Natl. Acad. Sci.,80:2026-2030 [1983]) and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New YorkN.Y., pp 77-96 [1985]).

[0209] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al. (Orlandi et al., Proc. Natl. Acad. Sci., 86: 3833 [1989];and Winter and Milstein, Nature 349:293 [1991]).

[0210] Antibody fragments which contain specific binding sites fortelomerase or telomerase protein subunits may also be generated. Forexample, such fragments include, but are not limited to, the F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab fragments which can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fabexpression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse et al., Science 256:1275 [1989]).

[0211] A variety of protocols for competitive binding orimmunoradiometric assays using either polyclonal or monoclonalantibodies with established specificities are well known in the art.Such immunoassays typically involve the formation of complexes betweentelomerase or telomerase protein subunit and its specific antibody andthe measurement of complex formation. A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twononinterfering epitopes on a specific telomerase protein subunit ispreferred in some situations, but a competitive binding assay may alsobe employed (See e.g., Maddox et al., J. Exp. Med., 158:1211 [1983]).

[0212] Peptides selected from the group comprising the sequences shownin FIG. 32 are used to generate polyclonal and monoclonal antibodiesspecifically directed against human and other telomerase proteins. Thepeptides are useful for inhibition of protein-RNA, protein-proteininteraction within the telomerase complex, and protein-DNA interactionat telomeres. Antibodies produced against these peptides are then usedin various settings, including but not limited to anti-cancertherapeutics capable of inhibiting telomerase activity, for purificationof native telomerase for therapeutics, for purification and cloningother components of human telomerase and other proteins associated withhuman telomerase, and diagnostic reagents.

[0213] Diagnostic Assays Using Telomerase Specific Antibodies

[0214] Particular telomerase and telomerase protein subunit antibodiesare useful for the diagnosis of conditions or diseases characterized byexpression of telomerase or telomerase protein subunits, or in assays tomonitor patients being treated with telomerase, its fragments, agonistsor inhibitors (including antisense transcripts capable of reducingexpression of telomerase). Diagnostic assays for telomerase includemethods utilizing the antibody and a label to detect telomerase in humanbody fluids or extracts of cells or tissues. The polypeptides andantibodies of the present invention may be used with or withoutmodification. Frequently, the polypeptides and antibodies will belabeled by joining them, either covalently or noncovalently, with areporter molecule. A wide variety of reporter molecules are known,several of which were described above. In particular, the presentinvention is useful for diagnosis of human disease, although it iscontemplated that the present invention will find use in the veterinaryarena.

[0215] A variety of protocols for measuring telomerase protein(s) usingeither polyclonal or monoclonal antibodies specific for the respectiveprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson the telomerase proteins or a subunit is preferred, but a competitivebinding assay may be employed. These assays are described, among otherplaces, in Maddox (Maddox et al., J. Exp. Med., 158:1211 [1983]).

[0216] In order to provide a basis for diagnosis, normal or standardvalues for human telomerase expression must be established. This isaccomplished by combining body fluids or cell extracts taken from normalsubjects, either animal or human, with antibody to telomerase ortelomerase subunit(s) under conditions suitable for complex formationwhich are well known in the art. The amount of standard complexformation may be quantified by comparing various artificial membranescontaining known quantities of telomerase protein, with both control anddisease samples from biopsied tissues. Then, standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom subjects potentially affected by disease (e.g., metastases).Deviation between standard and subject values establishes the presenceof a disease state.

[0217] Drug Screening

[0218] Telomerase or telomerase subunit proteins or their catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening therapeutic compounds in any of a variety of drug screeningtechniques. The fragment employed in such a test may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweentelomerase or the subunit protein and the agent being tested, may bemeasured.

[0219] Another technique for drug screening which may be used for highthroughput screening of compounds having suitable binding affinity tothe telomerase or telomerase protein subunit is described in detail in“Determination of Amino Acid Sequence Antigenicity” by Geysen, (Geysen,WO Application 84/03564, published on Sep. 13, 1984, incorporated hereinby reference). In summary, large numbers of different small peptide testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The peptide test compounds are reacted withfragments of telomerase or telomerase protein subunits and washed. Boundtelomerase or telomerase protein subunit is then detected by methodswell known in the art. Substantially purified telomerase or telomeraseprotein subunit can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0220] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingtelomerase or subunit protein(s) specifically compete with a testcompound for binding telomerase or the subunit protein. In this manner,the antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with the telomerase or subunitprotein.

[0221] Uses of the Polynucleotides Encoding Telomerase Subunit Proteins

[0222] A polynucleotide sequence encoding telomerase subunit proteins orany part thereof may be used for diagnostic and/or therapeutic purposes.For diagnostic purposes, the sequence encoding telomerase subunitprotein of this invention may be used to detect and quantitate geneexpression of the telomerase or subunit protein. The diagnostic assay isuseful to distinguish between absence, presence, and excess expressionof telomerase, and to monitor regulation of telomerase levels duringtherapeutic intervention. Included in the scope of the invention areoligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.

[0223] Another aspect of the subject invention is to provide forhybridization or PCR probes which are capable of detectingpolynucleotide sequences, including genomic sequences, encodingtelomerase subunit proteins or closely related molecules. Thespecificity of the probe, whether it is made from a highly specificregion (e.g., 10 unique nucleotides in the 5′ regulatory region), or aless specific region (e.g., especially in the 3′ region), and thestringency of the hybridization or amplification (maximal, high,intermediate or low) will determine whether the probe identifies onlynaturally occurring telomerase, telomerase subunit proteins or relatedsequences.

[0224] Probes may also be used for the detection of related sequencesand should preferably contain at least 50% of the nucleotides from anyof these telomerase subunit protein sequences. The hybridization probesof the subject invention may be derived from the nucleotide sequenceprovided by the present invention (e.g., SEQ ID NO: 1, 3, 62, 66, or69), or from genomic sequence including promoter, enhancer elements andintrons of the naturally occurring sequence encoding telomerase subunitproteins. Hybridization probes may be labeled by a variety of reportergroups, including commercially available radionuclides such as ³²P or35S, or enzymatic labels such as alkaline phosphatase coupled to theprobe via avidin/biotin coupling systems, and the like.

[0225] Other means for producing specific hybridization probes for DNAsinclude the cloning of nucleic acid sequences encoding telomerasesubunit proteins or derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art and are commercially availableand may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerase as T7 or SP6 RNA polymeraseand the appropriate radioactively labeled nucleotides.

[0226] Diagnostic Use

[0227] Polynucleotide sequences encoding telomerase may be used for thediagnosis of conditions or diseases with which the abnormal expressionof telomerase is associated. For example, polynucleotide sequencesencoding human telomerase may be used in hybridization or PCR assays offluids or tissues from biopsies to detect telomerase expression. Theform of such qualitative or quantitative methods may include Southern ornorthern analysis, dot blot or other membrane-based technologies; PCRtechnologies; dip stick, pin, chip and ELISA technologies. All of thesetechniques are well known in the art and are the basis of manycommercially available diagnostic kits.

[0228] The human telomerase-encoding nucleotide sequences disclosedherein provide the basis for assays that detect activation or inductionassociated with disease (including metastasis); in addition, the lack ofexpression of human telomerase may be detected using the human and othertelomerase-encoding nucleotide sequences disclosed herein. Thenucleotide sequence may be labeled by methods known in the art and addedto a fluid or tissue sample from a patient under conditions suitable forthe formation of hybridization complexes. After an incubation period,the sample is washed with a compatible fluid which optionally contains adye (or other label requiring a developer) if the nucleotide has beenlabeled with an enzyme. After the compatible fluid is rinsed off, thedye is quantitated and compared with a standard. If the amount of dye inthe biopsied or extracted sample is significantly elevated over that ofa comparable control sample, the nucleotide sequence has hybridized withnucleotide sequences in the sample, and the presence of elevated levelsof nucleotide sequences encoding human telomerase in the sampleindicates the presence of the associated disease. Alternatively, theloss of expression of human telomerase sequences in a tissue whichnormally expresses telomerase sequences indicates the presence of anabnormal or disease state.

[0229] Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regime in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient. Inorder to provide a basis for the diagnosis of disease, a normal orstandard profile for human telomerase expression must be established.This is accomplished by combining body fluids or cell extracts takenfrom normal subjects, either animal or human, with human telomerase or aportion thereof, under conditions suitable for hybridization oramplification. Standard hybridization may be quantified by comparing thevalues obtained for normal subjects with a dilution series of humantelomerase run in the same experiment where a known amount ofsubstantially purified human telomerase is used. Standard valuesobtained from normal samples may be compared with values obtained fromsamples from patients affected by telomerase-associated diseases.Deviation between standard and subject values establishes the presenceof disease.

[0230] Once disease is established, a therapeutic agent is administeredand a treatment profile is generated. Such assays may be repeated on aregular basis to evaluate whether the values in the profile progresstoward or return to the normal or standard pattern. Successive treatmentprofiles may be used to show the efficacy of treatment over a period ofseveral days or several months.

[0231] PCR, which may be used as described in U.S. Pat. Nos. 4,683,195,4,683,202, and 4,965,188 (herein incorporated by reference) providesadditional uses for oligonucleotides based upon the sequence encodingtelomerase subunit proteins. Such oligomers are generally chemicallysynthesized, but they may be generated enzymatically or produced from arecombinant source. Oligomers generally comprise two nucleotidesequences, one with sense orientation (5→3′) and one with antisense(3←5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

[0232] Additionally, methods which may be used to quantitate theexpression of a particular molecule include radiolabeling (Melby et al.,J. Immunol. Meth., 159:235-44 [1993]) or biotinylating [Duplaa et al.,Anal. Biochem., 229-36 [1993]) nucleotides, co-amplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated. Quantitation of multiple samples may bespeeded up by running the assay in an ELISA format where the oligomer ofinterest is presented in various dilutions and a spectrophotometric orcolorimetric response gives rapid quantitation. A definitive diagnosisof this type may allow health professionals to begin aggressivetreatment and prevent further worsening of the condition. Similarly,further assays can be used to monitor the progress of a patient duringtreatment. Furthermore, the nucleotide sequences disclosed herein may beused in molecular biology techniques that have not yet been developed,provided the new techniques rely on properties of nucleotide sequencesthat are currently known such as the triplet genetic code, specific basepair interactions, and the like.

[0233] Therapeutic Use

[0234] Based upon its homology to other telomerase sequences, thepolynucleotide encoding human telomerase disclosed herein may be usefulin the treatment of metastasis; in particular, inhibition of humantelomerase expression may be therapeutic.

[0235] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences (sense or antisense) to the targetedorgan, tissue or cell population. Methods which are well known to thoseskilled in the art can be used to construct recombinant vectors whichwill express antisense of the sequence encoding human telomerase. See,for example, the techniques described in Sambrook et al (supra) andAusubel et al. (supra).

[0236] The polynucleotides comprising full length cDNA sequence and/orits regulatory elements enable researchers to use the sequence encodinghuman telomerase, including the various motifs as an investigative toolin sense (Youssoufian and Lodish, Mol. Cell. Biol., 13:98-104 [1993]) orantisense (Eguchi et al., Ann. Rev. Biochem., 60:631-652 [1991])regulation of gene function. Such technology is now well known in theart, and sense or antisense oligomers, or larger fragments, can bedesigned from various locations along the coding or control regions.

[0237] Genes encoding human telomerase can be turned off by transfectinga cell or tissue with expression vectors which express high levels of adesired telomerase fragment. Such constructs can flood cells withuntranslatable sense or antisense sequences. Even in the absence ofintegration into the DNA, such vectors may continue to transcribe RNAmolecules until all copies are disabled by endogenous nucleases.Transient expression may last for a month or more with a non-replicatingvector and even longer if appropriate replication elements are part ofthe vector system.

[0238] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA or PNA, to thecontrol regions of the sequence encoding human telomerase (i.e., thepromoters, enhancers, and introns). Oligonucleotides derived from thetranscription initiation site, (e.g., between −10 and +10 regions of theleader sequence) are preferred. The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes. Similarly, inhibition can be achieved using“triple helix” base-pairing methodology. Triple helix pairingcompromises the ability of the double helix to open sufficiently for thebinding of polymerases, transcription factors, or regulatory molecules(for a review of recent therapeutic advances using triplex DNA, see Geeet al., in Huber and Carr, Molecular and Immunologic Approaches, FuturaPublishing Co, Mt Kisco N.Y. [1994]).

[0239] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of the sequence encoding human telomerase.

[0240] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0241] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding human telomerase and/ortelomerase protein subunits. Such DNA sequences may be incorporated intoa wide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, antisense cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells or tissues.

[0242] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine and wybutosine as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0243] Methods for introducing vectors into cells or tissues includethose methods discussed infra, and which are equally suitable for invivo, in vitro and ex vivo therapy. For ex vivo therapy, vectors areintroduced into stem cells taken from the patient and clonallypropagated for autologous transplant back into that same patient ispresented in U.S. Pat. Nos. 5,399,493 and 5,437,994, the disclosure ofwhich is herein incorporated by reference. Delivery by transfection andby liposome are quite well known in the art.

[0244] Furthermore, the nucleotide sequences encoding the varioustelomerase proteins and subunits disclosed herein may be used inmolecular biology techniques that have not yet been developed, providedthe new techniques rely on properties of nucleotide sequences that arecurrently known, including but not limited to such properties as thetriplet genetic code and specific base pair interactions.

[0245] Detection and Mapping of Related Polynucleotide Sequences inOther Genomes

[0246] The nucleic acid sequence encoding E. aediculatus, S. cerevisiae,S. pombe , and human telomerase subunit proteins and sequence variantsthereof, may also be used to generate hybridization probes for mappingthe naturally occurring homologous genomic sequence in the human andother genomes. The sequence may be mapped to a particular chromosome orto a specific region of the chromosome using well known techniques.These include in situ hybridization to chromosomal spreads, flow-sortedchromosomal preparations, or artificial chromosome constructions such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial PI constructions or single chromosome cDNA libraries asreviewed by Price (Price, Blood Rev., 7:127 [1993]) and Trask (Trask,Trends Genet 7:149 [1991]).

[0247] The technique of fluorescent in situ hybridization (FISH) ofchromosome spreads has been described, among other places, in Verma etal (Verma et al., Human Chromosomes: A Manual of Basic Techniques,Pergamon Press, New York N.Y. [1988]). Fluorescent in situ hybridizationof chromosomal preparations and other physical chromosome mappingtechniques may be correlated with additional genetic map data. Examplesof genetic map data can be found in the 1994 Genome Issue of Science(265:1981f). Correlation between the location of the sequence encodinghuman telomerase on a physical chromosomal map and a specific disease(or predisposition to a specific disease) may help delimit the region ofDNA associated with the disease. The nucleotide sequences of the subjectinvention may be used to detect differences in gene sequences betweennormal, carrier or affected individuals.

[0248] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers are invaluable in extending genetic maps (See e.g.,Hudson et al., Science 270:1945 [1995]). Often the placement of a geneon the chromosome of another mammalian species such as mouse (WhiteheadInstitute/MIT Center for Genome Research, Genetic Map of the Mouse,Database Release 10, Apr. 28, 1995) may reveal associated markers evenif the number or arm of a particular human chromosome is not known. Newsequences can be assigned to chromosomal arms, or parts thereof, byphysical mapping. This provides valuable information to investigatorssearching for disease genes using positional cloning or other genediscovery techniques.

[0249] Pharmaceutical Compositions

[0250] The present invention also relates to pharmaceutical compositionswhich may comprise telomerase and/or or telomerase subunit nucleotides,proteins, antibodies, agonists, antagonists, or inhibitors, alone or incombination with at least one other agent, such as stabilizing compound,which may be administered in any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. Any of these molecules can be administered to apatient alone, or in combination with other agents, drugs or hormones,in pharmaceutical compositions where it is mixed with suitableexcipient(s), adjuvants, and/or pharmaceutically acceptable carriers. Inone embodiment of the present invention, the pharmaceutically acceptablecarrier is pharmaceutically inert.

[0251] Administration Of Pharmaceutical Compositions

[0252] Administration of pharmaceutical compositions is accomplishedorally or parenterally. Methods of parenteral delivery include topical,intra-arterial (e.g., directly to the tumor), intramuscular,subcutaneous, intramedullary, intrathecal, intraventricular,intravenous, intraperitoneal, or intranasal administration. In additionto the active ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers comprising excipients andother compounds that facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Maack Publishing Co,Easton Pa.).

[0253] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc.,suitable for ingestion by the patient.

[0254] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable additional compounds, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers include, but are not limited to sugars, including lactose,sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato,or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; as well as proteins such asgelatin and collagen. If desired, disintegrating or solubilizing agentsmay be added, such as the cross-linked polyvinyl pyrrolidone, agar,alginic acid, or a salt thereof, such as sodium alginate.

[0255] Dragee cores are provided with suitable coatings such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage).

[0256] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

[0257] Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

[0258] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0259] Manufacture And Storage

[0260] The pharmaceutical compositions of the present invention may bemanufactured in a manner that known in the art (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes).

[0261] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0262] After pharmaceutical compositions comprising a compound of theinvention formulated in a acceptable carrier have been prepared, theycan be placed in an appropriate container and labeled for treatment ofan indicated condition. For administration of human telomerase proteins,such labeling would include amount, frequency and method ofadministration.

[0263] Therapeutically Effective Dose

[0264] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose. Thedetermination of an effective dose is well within the capability ofthose skilled in the art.

[0265] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays or in any appropriateanimal model. The animal model is also used to achieve a desirableconcentration range and route of administration. Such information canthen be used to determine useful doses, and routes for administration inhumans.

[0266] A therapeutically effective dose refers to that amount of proteinor its antibodies, antagonists, or inhibitors which ameliorate thesymptoms or condition. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals (e.g., ED₅₀, the dosetherapeutically effective in 50% of the population; and LD₅₀, the doselethal to 50% of the population). The dose ratio between therapeutic andtoxic effects is the therapeutic index, and it can be expressed as theratio, LD₅/ED₅₀. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage of such compounds lies preferably within a rangeof circulating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0267] The exact dosage is chosen by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors which may be taken into accountinclude the severity of the disease state (e.g., tumor size andlocation; age, weight and gender of the patient; diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy). Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation. Guidance as to particular dosagesand methods of delivery is provided in the literature (See, U.S. Pat.Nos. 4,657,760; 5,206,344; and 5,225,212, herein incorporated byreference). Those skilled in the art will employ different formulationsfor nucleotides than for proteins or their inhibitors. Similarly,delivery of polynucleotides or polypeptides will be specific toparticular cells, conditions, locations, etc.

[0268] It is contemplated, for example, that human telomerase can beused as a therapeutic molecule combat disease (e.g., cancer) and/orproblems associated with aging. It is further contemplated thatantisense molecules capable of reducing the expression of humantelomerase or telomerase protein subunits can be as therapeuticmolecules to treat tumors associated with the aberrant expression ofhuman telomerase. Still further it is contemplated that antibodiesdirected against human telomerase and capable of neutralizing thebiological activity of human telomerase may be used as therapeuticmolecules to treat tumors associated with the aberrant expression ofhuman telomerase and/or telomerase protein subunits.

[0269] EXPERIMENTAL

[0270] The following examples are provided in order to demonstrate andfurther illustrate certain preferred embodiments and aspects of thepresent invention and are not to be construed as limiting the scopethereof.

[0271] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); 1 or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); μm (nanometers); ° C.(degrees Centigrade); RPN (ribonucleoprotein); remN(2′-O-methylribonucleotides); dNTP (deoxyribonucleotide); dH₂O(distilled water); DDT (dithiothreitol); PMSF (phenylmethylsulfonylfluoride); TE (10 mM Tris HCl, 1 mM EDTA, approximately pH 7.2); KGlu(potassium glutamate); SSC (salt and sodium citrate buffer); SDS (sodiumdodecyl sulfate); PAGE (polyacrylamide gel electrophoresis); Novex(Novex, San Diego, Calif.); BioRad (Bio-Rad Laboratories, Hercules,Calif.); Pharmacia (Pharmacia Biotech, Piscataway, N.J.);Boehringer-Mannheim (Boehringer-Mannheim Corp., Concord, Calif.);Amersham (Amersham, Inc., Chicago, Ill.); Stratagene (Stratagene CloningSystems, La Jolla, Calif.); NEB (New England Biolabs, Beverly, Mass.);Pierce (Pierce Chemical Co., Rockford, Ill.); Beckman (BeckmanInstruments, Fullerton, Calif.); Lab Industries (Lab Industries, Inc.,Berkeley, Calif.); Eppendorf (Eppendorf Scientific, Madison, Wis.); andMolecular Dynamics (Molecular Dynamics, Sunnyvale, Calif.).

EXAMPLE 1 Growth of Euplotes aediculatus

[0272] In this Example, cultures of E. aediculatus were obtained fromDr. David Prescott, MCDB, University of Colorado. Dr. Prescottoriginally isolated this culture from pond water, although this organismis also available from the ATCC (ATCC #30859). Cultures were grown asdescribed by Swanton et al., (Swanton et al., Chromosoma 77:203 [1980]),under non-sterile conditions, in 15-liter glass containers containingChlorogonium as a food source. Organisms were harvested from thecultures when the density reached approximately 10⁴ cells/ml.

EXAMPLE 2 Preparation of Nuclear Extracts

[0273] In this Example, nuclear extracts of E. aediculatus were preparedusing the method of Lingner et al., (Lingner et al., Genes Develop.,8:1984 [1994]), with minor modifications, as indicated below. Briefly,cells grown as described in Example 1 were concentrated with 15 μm Nytexfilters and cooled on ice. The cell pellet was resuspended in a finalvolume of 110 ml TMS/PMSF/spermidinephosphate buffer. The stockTMS/PMSF/spermidine phosphate buffer was prepared by adding 0.075 gspermidine phosphate (USB) and 0.75 ml PMSF (from 100 mM stock preparedin ethanol) to 150 ml TMS. TMS comprised 10 mM Tris-acetate, 10 mMMgCl₂, 85.5752 g sucrose/liter, and 0.33297 g CaCl₂/liter, pH 7.5.

[0274] After resuspension in TMS/PMSF/spermidinephosphate buffer, 8.8 ml10% NP-40 and 94.1 g sucrose were added and the mixture placed in asiliconized glass beaker with a stainless steel stirring rod attached toan overhead motor. The mixture was stirred until the cells werecompletely lysed (approximately 20 minutes). The mixture was thencentrifuged for 10 minutes at 7500 rpm (8950× g), at 4° C., using aBeckman JS-13 swing-out rotor. The supernatant was removed and nucleipellet was resuspended in TMS/PMSF/spermidine phosphate buffer, andcentrifuged again, for 5 minutes at 7500 rpm (8950× g), at 4° C., usinga Beckman JS-13 swing-out rotor.

[0275] The supernatant was removed and the nuclei pellet was resuspendedin a buffer comprised of 50 mM Tris-acetate, 10 mM MgCl₂, 10% glycerol,0.1% NP-40, 0.4 M KGlu, 0.5 mM PMSF, pH 7.5, at a volume of 0.5 mlbuffer per 10 g of harvested cells. The resuspended nuclei were thendounced in a glass homogenizer with approximately 50 strokes, and thencentrifuged for 25 minutes at 14,000 rpm at 4° C., in an Eppendorfcentrifuge. The supernatant containing the nuclear extract wascollected, frozen in liquid nitrogen, and stored at −80° C. until used.

EXAMPLE 3 Purification of Telomerase

[0276] In this Example, nuclear extracts prepared as described inExample 2 were used to purify E. aediculatus telomerase. In thispurification protocol, telomerase was first enriched by chromatographyon an Affi-Gel-heparin column, and then extensively purified by affinitypurification with an antisense oligonucleotide. As the template regionof telomerase RNA is accessible to hybridization in the telomerase RNPparticle, an antisense oligonucleotide (i.e., the “affinityoligonucleotide”) was synthesized that was complementary to thistemplate region as an affinity bait for the telomerase. A biotin residuewas included at the 5′ end of the oligonucleotide to immobilize it to anavidin column.

[0277] Following the binding of the telomerase to the oligonucleotide,and extensive washing, the telomerase was eluted by use of adisplacement oligonucleotide. The affinity oligonucleotide included DNAbases that were not complementary to the telomerase RNA 5′ to thetelomerase-specific sequence. As the displacement oligonucleotide wascomplementary to the affinity oligonucleotide for its entire length, itwas able to form a more thermodynamically stable duplex than thetelomerase bound to the affinity oligonucleotide. Thus, addition of thedisplacement oligonucleotide resulted in the elution of the telomerasefrom the column.

[0278] In this Example, the nuclear extracts prepared from 45 litercultures were frozen until a total of 34 ml of nuclear extract wascollected. This corresponded to 630 liters of culture (i.e.,approximately 4×10⁹ cells). The nuclear extract was diluted with abuffer to 410 ml, to provide final concentrations of 20 mM Tris-acetate,1 mM MgCl₂, 0.1 mM EDTA, 33 mM KGlu, 10% (vol/vol) glycerol, 1 mMdithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF),at a pH of 7.5.

[0279] The diluted nuclear extract was applied to an Affi-Gel-heparingel column (Bio-Rad), with a 230 ml bed volume and 5 cm diameter,equilibrated in the same buffer and eluted with a 2-liter gradient from33 to 450 mM KGlu. The column was run at 4° C., at a flow rate of 1column volume/hour. Fractions of 50 mls each were collected and assayedfor telomerase activity as described in Example 4. Telomerase was elutedfrom the column at approximately 170 mM KGlu. Fractions containingtelomerase (approximately 440 ml) were pooled and adjusted to 20 mMTris-acetate, 10 mM MgCl₂, 1 mM EDTA, 300 mM KGlu, 10% glycerol, 1 mMDTT, and 1% Nonidet P-40. This buffer was designated as “WB.”

[0280] To this preparation, 1.5 nmol of each of two competitor DNAoligonucleotides (5′-TAGACCTGTTAGTGTACATTTGAATTGAAGC-3′ (SEQ ID NO:28))and (5′-TAGACCTGTTAGGTTGGATTTGTGGCATCA-3′ (SEQ ID NO:29)), 50 μg yeastRNA (Sigma), and 0.3 nmol of biotin-labelled telomerase-specificoligonucleotide(5′-biotin-TAGACCTGTTA-(rmeG)₂-(rmeU)₄-(rmeG)₄-(rmeU)₄-remG-3 ′)(SEQ IDNO:60), were added per ml of the pool. The 2-O-methyribonucleotides ofthe telomerase specific oligonucleotides were complementary to thetelomerase RNA template region; the deoxyribonucleotides were notcomplementary. The inclusion of competitor, non-specific DNAoligonucleotides increased the efficiency of the purification, as theeffects of nucleic acid binding proteins and other components in themixture that would either bind to the affinity oligonucleotide or removethe telomerase from the mixture were minimized.

[0281] This material was then added to Ultralink immobilized neutravidinplus (Pierce) column material, at a volume of 60 μl of suspension per mlof pool. The column material was pre-blocked twice for 15 minutes eachblocking, with a preparation of WB containing 0.01% Nonidet P-40, 0.5 mgBSA, 0.5 mg/ml lysozyme, 0.05 mg/ml glycogen, and 0.1 mg/ml yeast RNA.The blocking was conducted at 4° C., using a rotating wheel tothoroughly block the column material. After the first blocking step, andbefore the second blocking step, the column material was centrifuged at200× g for 2 minutes to pellet the matrix.

[0282] The pool-column mixture was incubated for 8 minutes at 30° C.,and then for an additional 2 hours at 4° C., on a rotating wheel(approximately 10 rpm; Labindustries) to allow binding. The pool-columnmixture was then centrifuged 200×g for 2 minutes, and the supernatantcontaining unbound material was removed. The pool-column mixture wasthen washed. This washing process included the steps of rinsing thepool-column mixture with WB at 4° C., washing the mixture for 15 minuteswith WB at 4° C., rinsing with WB, washing for 5 minutes at 30° C., withWB containing 0.6 M KGlu, and no Nonidet P-40, washing 5 minutes at 25°C. with WB, and finally, rinsing again with WB. The volume remainingafter the final wash was kept small, in order to yield a ratio of bufferto column material of approximately 1:1.

[0283] Telomerase was eluted from the column material by adding 1 nmolof displacement deoxyoligonucleotide (5′-CA₄C₄A₄C₂TA₂CAG₂TCTA-3′)(SEQ IDNO:30), per ml of column material and incubating at 25° C. for 30minutes. The material was centrifuged for 2 minutes 14,000 rpm in amicrocentrifuge (Eppendorf), and the eluate collected. The elutionprocedure was repeated twice more, using fresh displacementoligonucleotide each time. As mentioned above, because the displacementoligonucleotide was complementary to the affinity oligonucleotide, itformed a more thermodynamically stable complex with the affinityoligonucleotide than the telomerase. Thus, addition of the displacementoligonucleotide to an affinity-bound telomerase resulted in efficientelution of telomerase under native conditions. The telomerase appearedto be approximately 50% pure at this stage, as judged by analysis on aprotein gel. The affinity purification of telomerase and elution with adisplacement oligonucleotide is shown in FIG. 1 (panels A and B,respectively). In this Figure, the 2′-O-methyl sugars of the affinityoligonucleotide are indicated by the bold line. The black and shadedoval shapes in this Figure are intended to graphically represent theprotein subunits of the present invention.

[0284] The protein concentrations of the extract and material obtainedfollowing Affi-Gel-heparin column chromatography, were determined usingthe method of Bradford (Bradford, Anal. Biochem., 72:248 [1976]), usingBSA as the standards. Only a fraction of the telomerase preparation wasfurther purified on a glycerol gradient.

[0285] The sedimentation coefficient of telomerase was determined byglycerol gradient centrifugation, as described in Example 8.

[0286] Table 1 below is a purification table for telomerase purifiedaccording to the methods of this Example. The telomerase was enriched12-fold in nuclear extracts, as compared to whole cell extracts, with arecovery of 80%; 85% of telomerase was solubilized from nuclei uponextraction. TABLE 1 Purification of Telomerase Telomerase Telomerase/(pmol of Protein/pmol Recovery Purification Fraction Protein (mg) RNP)of RNP/mg (%) Factor Nuclear 2020 1720 0.9 100 1 Extract Heparin 1251040 8.3 60 10 Affinity 0.3** 680 2270 40 2670 Glycerol NA* NA* NA* 25NA* Gradient

EXAMPLE 4 Telomerase Activity

[0287] At each step in the purification of telomerase, the preparationwas analyzed by three separate assays, one of which was activity, asdescribed in this Example. In general, telomerase assays were done in 40μl containing 0.003-0.3 ,μl of nuclear extract, 50 mM Tris-Cl (pH 7.5),50 mM KGlu, 10 mM MgCl₂, 1 mM DTT, 125 μM dTTP, 125 μM dGTP, andapproximately 0.2 pmoles of 5′-³²P-labelled oligonucleotide substrate(i.e., approximately 400,000 cpm). Oligonucleotide primers wereheat-denatured prior to their addition to the reaction mixture.Reactions were assembled on ice and incubated for 30 minutes at 25° C.The reactions were stopped by addition of 200 μl of 10 mM Tris-Cl (pH7.5), 15 mM EDTA, 0.6% SDS, and 0.05 mg/ml proteinase K, and incubatedfor at least 30 minutes at 45° C. After ethanol precipitation, theproducts were analyzed on denaturing 8% PAGE gels, as known in the art(See e.g., Sambrook et al., 1989).

EXAMPLE 5 Quantification of Telomerase Activity

[0288] In this Example, quantification of telomerase activity throughthe purification procedure is described. Quantitation was accomplishedby assaying the elongation of oligonucleotide primers in the presence ofdGTP and [α-³²P]dTTP. Briefly, 1 μM 5′-(G₄T₄)₂-3′ oligonucleotide wasextended in a 20 μl reaction mixture in the presence of 2 μl of[α-³²P]dTTP (10 mCi/ml, 400 Ci/mmol; 1 Ci=37 GBq), and 125 μM dGTP asdescribed by (Lingner et al., Genes Develop., 8:1984 [1994]), and loadedonto an 8% PAGE sequencing gel as known in the art (See e.g., Sambrooket al., 1989).

[0289] The results of this study are shown in FIG. 3. In lane 1, thereis no telomerase present (i.e., a negative control); lanes 2, 5, 8, and11 contained 0.14 fmol telomerase; lanes 3,6,9, and 12 contained 0.42fmol telomerase; and lanes 4, 7, 10, and 13 contained 1.3 fmoltelomerase. Activity was quantified using a Phosphorlmager (MolecularDynamics) using the manufacturer's instructions. It was determined thatunder these conditions, 1 fmol of affinity-purified telomeraseincorporated 21 fmol of dTTP in 30 minutes.

[0290] As shown in this figure, the specific activity of the telomerasedid not change significantly through the purification procedure.Affinity-purified telomerase was fully active. However, it wasdetermined that at high concentrations, an inhibitory activity wasdetected and the activity of crude extracts was not linear. Thus, in theassay shown in FIG. 3, the crude extract was diluted 700-7000-fold. Uponpurification, this inhibitory activity was removed and no inhibitoryeffect was detected in the purified telomerase preparations, even athigh enzyme concentrations.

EXAMPLE 6 Gel Electrophoresis and Northern Blots

[0291] As indicated in Example 4, at each step in the purification oftelomerase, the preparation was analyzed by three separate assays. ThisExample describes the gel electrophoresis and blotting procedures usedto quantify telomerase RNA present in fractions and analyze theintegrity of the telomerase ribonucleoprotein particle.

[0292] Denaturing Gels and Northern Blots

[0293] In this Example, synthetic T7-transcribed telomerase RNA of knownconcentration served as the standard. Throughout this investigation, theRNA component was used as a measure of telomerase.

[0294] A construct for phage T7 RNA polymerase transcription of E.aediculatus telomerase RNA was produced, using the polymerase chainreaction (PCR). The telomerase RNA gene was amplified with primers thatannealed to either end of the gene. The primer that annealed at the 5′end also encoded a hammerhead ribozyme sequence to generate the natural5′ end upon cleavage of the transcribed RNA, a T7-promoter sequence, andan EcoRI site for subcloning. The sequence of this 5′ primer was5′-GCGGGAATTCTAATACGACTCACTATAGGGAAGAAACTCTGATGAGGCCGAAAGGCCGAAACTCCACGAAAGTGGAGTAAGTTTCTCGATAATTGATCTGTAG-3′ (SEQ ID NO:31).The 3′ primer included an EarI site for termination of transcription atthe natural 3′ end, and a BamHI site for cloning. The sequence of this3′ primer was 5′-CGGGGATCCTCTTCAAAAGATGAGAGGACAGCAAAC-3′ (SEQ ID NO:32).The PCR amplification product was cleaved with EcoRI and BamHI, andsubcloned into the respective sites of pUC19 (NEB), to give “pEaT7.” Thecorrectness of this insert was confirmed by DNA sequencing. T7transcription was performed as described by Zaug et al., Biochemistry33:14935 [1994]), with EarI-linearized plasmid. RNA was gel-purified andthe concentration was determined (an A₂₆₀ of 1=40 μg/ml). This RNA wasused as a standard to determine the telomerase RNA present in variouspreparations of telomerase.

[0295] The signal of hybridization was proportional to the amount oftelomerase RNA, and the derived RNA concentrations were consistent with,but slightly higher than those obtained by native gel electrophoresis.Comparison of the amount of whole telomerase RNA in whole cell RNA toserial dilutions of known T7 RNA transcript concentrations indicatedthat each E. aediculatus cell contained approximately 300,000 telomerasemolecules.

[0296] Visualization of the telomerase was accomplished by Northern blothybridization to its RNA component, using the methods described byLingner et al. (Linger et al., Genes Develop., 8:1984 [1994]). Briefly,RNA (less than or equal to 0.5 μg/lane) was resolved on an 8% PAGE andelectroblotted onto a Hybond-N membrane (Amersham), as known in the art(See e.g., Sambrook et al., 1989). The blot was hybridized overnight in10 ml of 4× SSC, lOx Denhardt's solution, 0.1% SDS, and 50 μg/mldenatured herring sperm DNA,. After pre-hybridizing for 3 hours, 2×10⁶cpm probe/ml hybridization solution was added. The randomly labelledprobe was a PCR-product that covered the entire telomerase RNA gene. Theblot was washed with several buffer changes for 30 minutes in 2× SSC,0.1% SDS, and then washed for 1 hour in 0.1× SSC and 0.1% SDS at 45° C.

[0297] Native Gels and Northern Blots

[0298] In this experiment, the purified telomerase preparation was runon native (i.e., non-denaturing) gels of 3.5% polyacrylamide and 0.33%agarose, as known in the art and described by Lamond and Sproat (Lamondand Sproat, [1994], supra). The telomerase comigrated approximately withthe xylene cyanol dye.

[0299] The native gel results indicated that telomerase was maintainedas an RNP throughout the purification protocol. FIG. 2 is a photographof a Northern blot showing the mobility of the telomerase in differentfractions on a non-denaturing gel as well as in vitro transcribedtelomerase. In this figure, lane 1 contained 1.5 fmol telomerase RNA,lane 2 contained 4.6 fmol telomerase RNA, lane 3 contained 14 fmoltelomerase RNA, lane 4 contained 41 fmol telomerase RNA, lane 5contained nuclear extract (42 fmol telomerase), lane 6 containedAffi-Gel-heparin-purified telomerase (47 fmol telomerase), lane 7contained affinity-purified telomerase (68 fmol), and lane 8 containedglycerol gradient-purified telomerase (35 fmol).

[0300] As shown in FIG. 2, in nuclear extracts, the telomerase wasassembled into an RNP particle that migrated slower than unassembledtelomerase RNA. Less than 1% free RNA was detected by this method.However, a slower migrating telomerase RNP complex was also sometimesdetected in extracts. Upon purification on the Affi-Gel-heparin column,the telomerase RNP particle did not change in mobility (FIG. 2, lane 6).However, upon affinity purification the mobility of the RNA particleslightly increased (FIG. 2, lane 7), perhaps indicating that a proteinsubunit or fragment had been lost. On glycerol gradients, theaffinity-purified telomerase did not change in size, but approximately2% free telomerase RNA was detectable (FIG. 2, lane 8), suggesting thata small amount of disassembly of the RNP particle had occurred.

EXAMPLE 7 Telomerase Protein Composition

[0301] In this Example, the analysis of the purified telomerase proteincomposition are described.

[0302] In this Example, glycerol gradient fractions obtained fromExample 8, were separated on a 4-20% polyacrylamide gel (Novex).Following electrophoresis, the gel was stained with Coomassie brilliantblue. FIG. 4 shows a photograph of the gel. Lanes 1 and 2 containedmolecular mass markers (Pharmacia) as indicated on the left side of thegel shown in FIG. 4. Lanes 3-5 contained glycerol gradient fractionpools as indicated on the top of the gel (i.e., lane 3 containedfractions 9-14, lane 4 contained fractions 15-22, and lane 5 containedfractions 23-32). Lane 4 contained the pool with 1 pmol of telomeraseRNA. In lanes 6-9 BSA standards were run at concentrations indicated atthe top of the gel in FIG. 4 (i.e., lane 6 contained 0.5 pmol BSA, lane7 contained 1.5 pmol BSA, lane 8 contained 4.5 BSA, and lane 9 contained15 pmol BSA).

[0303] As shown in FIG. 4, polypeptides with molecular masses of 120 and43 kDa co-purified with the telomerase. The 43 kDa polypeptide wasobserved as a doublet. It was noted that the polypeptide ofapproximately 43 kDa in lane 3 migrated differently than the doublet inlane 4; it may be an unrelated protein. The 120 kDa and 43 kDa doubleteach stained with Coomassie brilliant blue at approximately the level of1 pmol, when compared with BSA standards. Because this fractioncontained 1 pmol of telomerase RNA, all of which was assembled into anRNP particle (See, FIG. 2, lane 8), there appear to be two polypeptidesubunits that are stoichiometric with the telomerase RNA. However, it isalso possible that the two proteins around 43 kDa are separate enzymesubunit.s

[0304] Affinity-purified telomerase that was not subjected tofractionation on a glycerol gradient contained additional polypeptideswith apparent molecular masses of 35 and 37 kDa, respectively. Thislatter fraction was estimated to be at least 50% pure. However, the 35kDa and 37 kDa polypeptides that were present in the affinity-purifiedmaterial were not reproducibly separated by glycerol gradientcentrifugation. These polypeptides may be contaminants, as they were notvisible in all activity-containing preparations.

EXAMPLE 8 Sedimentation Coefficient

[0305] The sedimentation coefficient for telomerase was determined byglycerol gradient centrifugation. In this Example, nuclear extract andaffinity-purified telomerase were fractionated on 15-40% glycerolgradients containing 20 mM Tris-acetate, with 1 mM MgCl₂, 0.1 mM EDTA,300 mM KGlu, and 1 mM DTT, at pH 7.5. Glycerol gradients were poured in5 ml (13×51 mm) tubes, and centrifuged using an SW55Ti rotor (Beckman)at 55,000 rpm for 14 hours at 4° C.

[0306] Marker proteins were run in a parallel gradient and had asedimentation coefficient of 7.6 S for alcohol dehydrogenase (ADH), 113S for catalase, 17.3 S for apoferritin, and 19.3 S for thyroglobulin.The telomerase peak was identified by native gel electrophoresis ofgradient fractions followed by blot hybridization to its RNA component.

[0307]FIG. 5 is a graph showing the sedimentation coefficient fortelomerase. As shown in this Figure, affinity-purified telomeraseco-sedimented with catalase at 11.5 S, while telomerase in nuclearextracts sedimented slightly faster, peaking around 12.5 S. Therefore,consistent with the mobility of the enzyme in native gels, purifiedtelomerase appears to have lost a proteolytic fragment or a looselyassociated subunit.

[0308] The calculated molecular mass for telomerase, if it is assumed toconsist of one 120 kDa protein subunit, one 43 kDa subunit, and one RNAsubunit of 66 kDa, adds up to a total of 229 kDa. This is in closeagreement with the 232 kDa molecular mass of catalase. However, thesedimentation coefficient is a function of the molecular mass, as wellas the partial specific volume and the frictional coefficient of themolecule, both of which are unknown for the telomerase RNP.

EXAMPLE 9 Substrate Utilization

[0309] In this Example, the substrate requirements of telomerase wereinvestigated. One simple model for DNA end replication predicts thatafter semi-conservative DNA replication, telomerase extendsdouble-stranded, blunt-ended DNA molecules. In a variation of thismodel, a single-stranded 3′ end is created by a helicase or nucleaseafter replication. This 3′ end is then used by telomerase for bindingand extension.

[0310] To determine whether telomerase is capable of elongatingblunt-ended molecules, model hairpins were synthesized with telomericrepeats positioned at their 3′ ends. These primer substrates weregel-purified, 5′-end labelled with polynucleotide kinase, heated at 0.4μM to 80° C. for 5 minutes, and then slowly cooled to room temperaturein a heating block, to allow renaturation and helix formation of thehairpins. Substrate mobility on a non-denaturing gel indicated that veryefficient hairpin formation was present, as compared to dimerization.

[0311] In this Example, assays were performed with unlabelled 125 μMdGTP, 125 μM dTTP, and 0.02 μM 5′-end-labelled primer (5′-³²P-labelledoligonucleotide substrate) in 10 μl reaction mixtures that contained 20mM Tris-acetate, with 10 mM MgCl₂, 50 mM KGlu, and 1 mM DTT, at pH 7.5.These mixtures were incubated at 25° C. for 30 minutes. Reactions werestopped by adding formamide loading buffer (i.e., TBE, formamide,bromthymol blue, and cyanol, Sambrook, 1989, supra).

[0312] Primers were incubated without telomerase (“−”), with 5.9 fmol ofaffinity-purified telomerase (“+”), or with 17.6 fmol ofaffinity-purified telomerase (“+++”). Affinity-purified telomerase usedin this assay was dialyzed with a membrane having a molecular cut-off of100 kDa, in order to remove the displacement oligonucleotide. Reactionproducts were separated on an 8% PAGE/urea gel containing 36% formamide,to denature the hairpins. The sequences of the primers used in thisstudy, as well as their lane assignments are shown in Table 2. TABLE 2Primer Sequences Lane Primer Sequence (5′ to 3′) SEQ ID NO:  1-3C₄(A₄C₄)₃CACA(G₄T₄)₃G₄ SEQ ID NO:33  4-6 C₂(A₄C₄)₃CACA(G₄T₄)₃G₄ SEQ IDNO:34  7-9 (A₄C₄)₃CACA(G₄T₄)₃G₄ SEQ ID NO:35 10-12A₂C₄(A₄C₄)₂CACA(G₄T₄)₃G₄ SEQ ID NO:36 13-15 C₄(A₄C₄)₂CACA(G₄T₄)₃ SEQ IDNO:37 16-18 (A₄C₄)₃CACA(G₄T₄)₃ SEQ ID NO:38 19-21 A₂C₄(A₄C₄)₂CACA(G₄T₄)₃SEQ ID NO:39 22-24 C₄(A₄C₄)₂CACA(G₄T₄)₃ SEQ ID NO:40 25-27C₂(A₄C₄)₂CACA(G₄T₄)₃ SEQ ID NO:41 28-30 (A₄C₄)₂CACA(G₄T₄)₃ SEQ ID NO:42

[0313] The gel results are shown in FIG. 6. Lanes 1-15 containedsubstrates with telomeric repeats ending with four G residues. Lanes16-30 contained substrates with telomeric repeats ending with four Tresidues. The putative alignment on the telomerase RNA template isindicated in FIG. 7 (SEQ ID NOS:43 and 44, and 45 and 46, respectively).It was assumed that the primer sets anneal at two very differentpositions in the template shown in FIG. 7 (i.e., 7A and 7B,respectively). This may have affected their binding and/or elongationrate.

[0314]FIG. 8 shows a lighter exposure of lanes 25-30 in FIG. 6. Thelighter exposure of FIG. 8 was taken in order to permit visualization ofthe nucleotides that are added and the positions of pausing in elongatedproducts. Percent of substrate elongated for the third lane in each setwas quantified on a Phosphorlmager, as indicated on the bottom of FIG.6.

[0315] The substrate efficiencies for these hairpins were compared withdouble-stranded telomere-like substrates with overhangs of differinglengths. A model substrate that ended with four G residues (see lanes1-15 of FIG. 6), was not elongated when it was blunt ended (see lanes1-3). However, slight extension was observed with an overhang length oftwo bases; elongation became efficient when the overhang was at least 4bases in length. The telomerase acted in a similar manner with adouble-stranded substrate that ended with four T residues, with a 6-baseoverhang required for highly efficient elongation. In FIG. 6, the faintbands below the primers in lanes 10-15 that are independent oftelomerase represent shorter oligonucleotides in the primerpreparations.

[0316] The lighter exposure of lanes 25-30 in FIG. 8 shows a ladder ofelongated products, with the darkest bands correlating with the putative5′ boundary of the template (as described by Lingner et al., GenesDevelop., 8:1984 [1994]). The abundance of products that correspond toother positions in the template suggested that pausing and/ordissociation occurs at sites other than the site of translocation withthe purified telomerase.

[0317] As shown in FIG. 6, double-stranded, blunt-ended oligonucleotideswere not substrates for telomerase. To determine whether these moleculeswould bind to telomerase, a competition experiment was performed. Inthis experiment, 2 nM of 5′-end labelled substrate with the sequence(G₄T₄)₂ (SEQ ID NO:61), or a hairpin substrate with a six base overhangrespectively were extended with 0.125 nM telomerase (FIG. 6, lanes25-27). Although the same unlabeled oligonucleotide substrates competedefficiently with labelled substrate for extension, no reduction ofactivity was observed when the double-stranded blunt-ended hairpinoligonucleotides were used as competitors, even in the presence of100-fold excess hairpins.

[0318] These results indicated that double-stranded, blunt-endedoligonucleotides cannot bind to telomerase at the concentrations testedin this Example. Rather, a single-stranded 3′ end is required forbinding. It is likely that this 3′ end is required to base pair with thetelomerase RNA template.

EXAMPLE 10 Cloning & Sequencing of the 123 kDa Polypeptide

[0319] In this Example, the cloning of the 123 kDa polypeptide oftelomerase (i.e., the 123 kDa protein subunit) is described. In thisstudy, an internal fragment of the telomerase gene was amplified by PCR,with oligonucleotide primers designed to match peptide sequences thatwere obtained from the purified polypeptide obtained in Example 3,above. The polypeptide sequence was determined using the nanoES tandemmass spectroscopy methods known in the art and described by Calvio etal., RNA 1:724-733 [1995]). The oligonucleotide primers used in thisExample had the following sequences, with positions that were degenerateshown in parentheses5′-TCT(G/A)AA(G/A)TA(G/A)TG(T/G/A)GT(G/A/T/C)A(T/G/A)(G/A)TT(G/A)TTCAT-3′(SEQ ID NO:47), AND5′-GCGGATCCATGAA(T/C)CC(A/T)GA(G/A)AA(T/C)CC(A/T)AA(T/C)GT-3′ (SEQ IDNO:48).

[0320] A 50 μl reaction contained 0.2 mM dNTPs, 0.15 μg E. aediculatuschromosomal DNA, 0.5 μl Taq (Boehringer-Mannheim), 0.8 μg of eachprimer, and lx reaction buffer (Boehringer-Mannheim). The reaction wasincubated in a thermocycler (Perkin-Elmer), using the following—5minutes at 95° C., followed by 30 cycles of 1 minute at 94° C., 1 minuteat 52° C., and 2 minutes at 72° C. The reaction was completed by 10minute incubation at 72° C.

[0321] A genomic DNA library was prepared from the chromosomal E.aediculatus DNA by cloning blunt-ended DNA into the SmaI site ofpCR-Script plasmid vector (Stratagene). This library was screened bycolony hybridization, with the radiolabelled, gel-purified PCR product.Plasmid DNA of positive clones was prepared and sequenced by the dideoxymethod (Sanger et al., Proc. Natl. Acad. Sci., 74:5463 [1977]) ormanually, through use of an automated sequencer (ABI). The DNA sequenceof the gene encoding this polypeptide is shown in FIG. 9 (SEQ ID NO:1).The start codon in this sequence inferred from the DNA sequence, islocated at nucleotide position 101, and the open reading frame ends atposition 3193. The genetic code of Euplotes differs from other organismsin that the “UGA” codon encodes a cysteine residue. The amino acidsequence of the polypeptide inferred from the DNA sequence is shown inFIG. 10 (SEQ ID NO:2), and assumes that no unusual amino acids areinserted during translation and no post-translational modificationoccurs.

EXAMPLE 11 Cloning & Sequencing of the 43 kDa Polypeptide

[0322] In this Example, the cloning of the 43 kDa polypeptide oftelomerase (i.e., the 43 kDa protein subunit) is described. In thisstudy, an internal fragment of the telomerase gene was amplified by PCR,with oligonucleotide primers designed to match peptide sequences thatwere obtained from the purified polypeptide obtained in Example 3,above. The polypeptide sequence was determined using the nanoES tandemmass spectroscopy methods known in the art and described by Calvio etal., RNA 1:724-733 [1995]). The oligonucleotide primers used in thisExample had the following sequences—5′-NNNGTNAC(C/T/A)GG(C/T/A)AT(C/T/A)AA(C/T)AA-3′ (SEQ ID NO:49), and5′-(T/G/A)GC(T/G/A)GT(C/T)TC(T/C)TG(G/A)TC(G/A)TT(G/A)TA-3′ (SEQ IDNO:50). In this sequence, “N” indicates the presence of any of the fournucleotides (i.e., A, T, G, or C).

[0323] A 50 μl reaction contained 0.2 mM dNTPs, 0.2 μg E. aediculatuschromosomal DNA, 0.5 μl Taq (Boehringer-Mannheim), 0.8 μg of eachprimer, and 1× reaction buffer (Boehringer-Mannheim). The reaction wasincubated in a thermocycler (Perkin-Elmer), using the following—5minutes at 95° C., followed by 30 cycles of 1 minute at 94° C., 1 minuteat 52° C., and 1 minutes at 72° C. The reaction was completed by 10minute incubation at 72° C.

[0324] A genomic DNA library was prepared from the chromosomal E.aediculatus DNA by cloning blunt-ended DNA into the SmaI site ofpCR-Script plasmid vector (Stratagene). This library was screened bycolony hybridization, with the radiolabelled, gel-purified PCR product.Plasmid DNA of positive clones was prepared and sequenced by the dideoxymethod (Sanger et al., Proc. Natl. Acad. Sci., 74:5463 [1977]) ormanually, through use of an automated sequencer (ABI). The DNA sequenceof the gene encoding this polypeptide is shown in FIG. 11 (SEQ ID NO:3).Three potential reading frames are shown for this sequence, as shown inFIG. 12. For clarity, the amino acid sequence is indicated below thenucleotide sequence in all three reading frames. These reading framesare designated as “a,” “b,” and “c” (SEQ ID NOS:4-6). A possible startcodon is encoded at nucleotide position 84 in reading frame “c.” Theycoding region could end at position 1501 in reading frame “b.” Earlystop codons, indicated by asterisks in this figure, occur in all threereading frames between nucleotide position 337-350.

[0325] The “La-domain” is indicated in bold-face type. Furtherdownstream, the protein sequence appears to be encoded by differentreading frames, as none of the three frames is uninterrupted by stopcodons. Furthermore, peptide sequences from purified protein are encodedin all three frames. Therefore, this gene appears to contain interveningsequences, or in the alternative, the RNA is edited. Other possibilitiesinclude ribosomal frame-shifting or sequence errors. However, thehomology to the La-protein sequence remains of significant interest.Again, in Euplotes, the “UGA” codon encodes a cysteine residue.

EXAMPLE 12 Amino Acid and Nucleic Acid Comparisons

[0326] In this Example, comparisons between various reported sequencesand the sequences of the 123 kDa and 43 kDa telomerase subunitpolypeptides were made.

[0327] Comparisons with the 123 kDa E. aediculatus Telomerase Subunit

[0328] The amino acid sequence of the 123 kDa Euplotes aediculatuspolypeptide was compared with the sequence of the 80 kDa telomeraseprotein subunit of Tetrahymena thermophila (GenBank accession #U25641)in order to investigate their similarity. The nucleotide sequence asobtained from GenBank (SEQ ID NO:51) encoding this protein is shown inFIG. 19. The amino acid sequence of this protein as obtained fromGenBank (SEQ ID NO:52) is shown in FIG. 20. The sequence comparisonbetween the 123 kDa E. aediculatus and 80 kDa T thermophila is shown inFIG. 13. In this figure, the E. aediculatus sequence is the uppersequence (SEQ ID NO:2), while the T. thermophila sequence is the lowersequence (SEQ ID NO:52). In this Figure, as well as FIGS. 14-16,identities are indicated by vertical bars, while single dots between thesequences indicate somewhat similar amino acids, and double dots betweenthe sequences indicate more similar amino acids. The observed identitywas determined to be approximately 19%, while the percent similarity wasapproximately 45%, values similar to what would be observed with anyrandom protein sequence.

[0329] The amino acid sequence of the 123 kDa Euplotes aediculatuspolypeptide was also compared with the sequence of the 95 kDa telomeraseprotein subunit of Tetrahymena thermophila (GenBank accession #U25642),in order to investigate their similarity. The nucleotide sequence asobtained from GenBank (SEQ ID NO:53) encoding this protein is shown inFIG. 21. The amino acid sequence of this protein as obtained fromGenBank (SEQ ID NO:54) is shown in FIG. 22. This sequence comparison isshown in FIG. 14. In this figure, the E. aediculatus sequence is theupper sequence (SEQ ID NO:2), while the T thermophila sequence is thelower sequence (SEQ ID NO:54); identities are indicated by verticalbars. The observed identity was determined to be approximately 20%,while the percent similarity was approximately 43%, values similar towhat would be observed with any random protein sequence.

[0330] Significantly, the amino acid sequence of the 123 kDa E.aediculatus polypeptide contains the five motifs (SEQ ID NOS:13 and 18)characteristic of reverse transcriptases. The 123 kDa polypeptide wasalso compared with the polymerase domains of various reversetranscriptases (SEQ ID NOS:14-17, and 19-22). FIG. 17 shows thealignment of the 123 kDa polypeptide with the putative yeast homolog(L8543.12 or ESTp)(SEQ ID NOS: 17 and 22). The amino acid sequence ofL8543.12 (or ESTp) obtained from GenBank is shown in FIG. 23 (SEQ IDNO:55).

[0331] Four motifs (A, B, C, and D) were included in this comparison. Inthis FIG. 17, highly conserved residues are indicated by white letterson a black background. Residues of the E. aediculatus sequences that areconserved in the other sequence are indicated in bold; the “h” indicatesthe presence of a hydrophobic amino acid. The numerals located betweenamino acid residues of the motifs indicates the length of gaps in thesequences. For example, the “100” shown between motifs A and B reflectsa 100 amino acid gap in the sequence between the motifs.

[0332] Genbank searches identified a yeast protein (Genbank accession#u20618), and gene “L8543.12” (Est2), containing amino acid sequencethat shows some homology to the E. aediculatus 123 kDa telomerasesubunit. Based on the observations that both proteins contain reversetranscriptase motifs in their C-terminal regions; both proteins sharesimilarity in regions outside the reverse transcriptase motif; theproteins are similarly basic (pI=10.1 for E. aediculatus and pI=10.0 forthe yeast); and both proteins are large (123 kDa for E. aediculatus and103 kDa for the yeast), these sequences comprise the catalytic core oftheir respective telomerases. It is contemplated that based on thisobservation of homology in two phylogenetically distinct organisms as E.aediculatus and yeast, the human telomerase will contain a protein thathas the same characteristics (i.e., reverse transcriptase motifs, isbasic, and large [>100 kDa]).

[0333] Comparisons with the 43 kDa E. aediculatus Telomerase Subunit

[0334] The amino acid sequence of the “La-domain” of the 43 kDa Euplotesaediculatus polypeptide was compared with the sequence of the 95 kDatelomerase protein subunit of Tetrahymena thermophila (described above)in order to investigate their similarity. This sequence comparison isshown in FIG. 15. In this figure, the E. aediculatus sequence is theupper sequence (SEQ ID NO:9), while the T. thermophila sequence is thelower sequence (SEQ ID NO:10); identities are indicated by verticalbars. The observed identity was determined to be approximately 23%,while the percent similarity was approximately 46%, values similar towhat would be observed with any random protein sequence.

[0335] The amino acid sequence of the “La-domain” of the 43 kDa Euplotesaediculatus polypeptide was compared with the sequence of the 80 kDatelomerase protein subunit of Tetrahymena thermophila (described above)in order to investigate their similarity. This sequence comparison isshown in FIG. 16. In this figure, the E. aediculatus sequence is theupper sequence (SEQ ID NO:11), while the T thermophila sequence is thelower sequence (SEQ ID NO:12); identities are indicated by verticalbars. The observed identity was determined to be approximately 26%,while the percent similarity was approximately 49%, values similar towhat would be observed with any random protein sequence.

[0336] The amino acid sequence of a domain of the 43 kDa E. aediculatuspolypeptide (SEQ ID NO:23) was also compared with La proteins fromvarious other organisms (SEQ ID NOS:24-27). These comparisons are shownin FIG. 18. In this Figure, highly conserved residues are indicated bywhite letters on a black background. Residues of the E. aediculatussequences that are conserved in the other sequence are indicated inbold.

EXAMPLE 13 Identification of Telomerase Protein Subunits in AnotherOrganism

[0337] In this Example, the sequences identified in the previousExamples above, were used to identify the telomerase protein subunits ofOxytricha trifallax, a ciliate that is very distantly related to E.aediculatus . In this Example, primers were chosen based on theconserved region of the E. aediculatus 123 kDa polypeptide whichcomprised the reverse transcriptase domain motifs. Suitable primers weresynthesized and used in a PCR reaction with total DNA from Oxytricha.The Oxytricha DNA was prepared according to methods known in the art.The PCR products were then cloned and sequenced using methods known inthe art.

[0338] The oligonucleotide sequences used as the primers were asfollows:5′-(T/C)A(A/G)AC(T/A/C)AA(G/A)GG(T/A/C)AT(T/C)CC(C/T/A)(C/T)A(G/A)GG-3′(SEQ ID NO:56) and5′-(G/A/T)GT(G/A/T)ATNA(G/A)NA(G/A)(G/A)TA(G/A)TC(G/A)TC-3′ (SEQ IDNO:57). Positions that were degenerate are shown in parenthesis, withthe alternative bases shown within the parenthesis. “N” represents anyof the four nucleotides.

[0339] In the PCR reaction, a 50 μl reaction contained 0.2 mM dNTPs, 0.3μg Oxytricha trifallax chromosomal DNA, 1 μl Taq polymerase(Boehringer-Mannheim), 2 micromolar of each primer, 1× reaction buffer(Boehringer-Mannheim). The reaction was incubated in a thermocycler(Perkin-Elmer) under the following conditions: 1×5 min at 95° C., 30cycles consisting of 1 min at 94° C., 1 min at 53° C., and 1 min at 72°C., followed by 1×10 min at 72° C. The PCR-product was gel-purified andsequenced by the dideoxy-method, by methods known well in the art (e.g.,Sanger et al., Proc. Natl. Acad. Sci. 74, 5463-5467 (1977).

[0340] The deduced amino acid sequence of the PCR product was determinedand compared with the E. aediculatus sequence. FIG. 24 shows thealignment of these sequences, with the O. trifallax sequence (SEQ IDNO:58) shown in the top row, and the E. aediculatus sequence (SEQ IDNO:59) shown in the bottom row. As can be seen from this Figure, thereis a great deal of homology between the O. trifallax polypeptidesequence identified in this Example with the E. aediculatus polypeptidesequence. Thus, it is clear that the sequences identified in the presentinvention are useful for the identification of homologous telomeraseprotein subunits in other eukaryotic organisms. Indeed, development ofthe present invention has identified homologous telomerase sequences inmultiple, diverse species.

EXAMPLE 15 Identification of Tetrahymena Telomerase Sequences

[0341] In this Example, a Tetrahymena clone was produced that shareshomology with the Euplotes sequences, and EST2p.

[0342] This experiment utilized PCR with degenerate oligonucleotideprimers directed against conserved motifs to identify regions ofhomology between Tetrahymena, Euplotes, and EST2p sequences. The PCRmethod used in this Example is a novel method that is designed tospecifically amplify rare DNA sequences from complex mixtures. Thismethod avoids the problem of amplification of DNA products with the samePCR primer at both ends (i.e., single primer products) commonlyencountered in PCR cloning methods. These single primer products produceunwanted background and can often obscure the amplification anddetection of the desired two-primer product. The method used in theseexperiment preferentially selects for two-primer products. Inparticular, one primer is biotinylated and the other is not. Afterseveral rounds of PCR amplification, the products are purified usingstreptavidin magnetic beads and two primer products are specificallyeluted using heat denaturation. This method finds use in settings otherthan the experiments described in this Example. Indeed, this methodfinds use in application in which it is desired to specifically amplifyrare DNA sequences, including the preliminary steps in cloning methodssuch as 5′ and 3; RACE, and any method that uses degenerate primers inPCR.

[0343] A first PCR run was conducted using Tetrahymena templatemacronuclear DNA isolated using methods known in the art, and the 24-merforward primer with the sequence 5′ biotin-GCCTATTT(TC)TT(TC)TA(TC)(GATC)(GATC)(GATC)AC(GATC)GA-3′ (SEQ ID NO:70) designated as“K231,” corresponding to the FFYXTE region (SEQ ID NO:71), and the23-mer reverse primer with the sequence5′-CCAGATAT(GATC)A(TGA)(GATC)A(AG)(AG)AA(AG)TC(AG)TC-3′ (SEQ ID NO:72),designated as “K220,” corresponding to the DDFL(FIL)I region (SEQ IDNO:73). This PCR reaction contained 2.5 μl DNA (50 ng), 4 μl of eachprimer (20 μM), 3 μl 10× PCR buffer, 3 μl 10× dNTPs, 2 μl Mg, 0.3 μlTaq, and 11.2 μl dH₂O. The mixture was cycled for 8 cycles of 94° C. for45 seconds, 37° C. for 45 seconds, and 72° C. for 1 minute.

[0344] This PCR reaction was bound to 200 μl streptavidin magneticbeads, washed with 200 μl TE, resuspended in 20 μl dH₂O and thenheat-denatured by boiling at 100° C. for 2 minutes. The beads werepulled down and the eluate removed. Then, 2.5 μl of this eluate wassubsequently reamplified using the above conditions, with the exceptionbieng that 0.3 μl of α-³²P DATP was included, and the PCR was carriedout for 33 cycles. This reaction was run a 5% denaturing polyacrylamidegel, and the appropriate region was cut out of the gel. These productswere then reamplified for an additional 34 cycles, under the conditionslisted above, with the exception being that a 42° C. annealingtemperature was used.

[0345] A second PCR run was conducted using Tetrahymena macronuclear DNAtemplate isolated using methods known in the art, and the 23-mer forwardprimer with the sequence 5′ACAATG(CA)G(GATC)(TCA)T(GATC)(TCA)T(GATC)CC(GATC)AA(AG)AA-3′ (SEQ IDNO:74), designated as “K228,” corresponding to the region R(LI)(LI)PKK(SEQ ID NO:75), and a reverse primer with the sequence5′-ACGAATC(GT)(GATC)GG(TAG)AT(GATC)(GC)(TA)(AG)TC(AG)TA(AG)CA 3′ (SEQ IDNO:76), designated “K224,” corresponding to the CYDSIPR region (SEQ IDNO:77). This PCR reaction contained 2.5 μl DNA (50 ng), 4 μl of eachprimer (20 μM), 3 μl 10× PCR buffer, 3 μl 10× dNTPs, 2 μl Mg, 0.3 μl a_(—) ³²P DATP, 0.3 μl Taq, and 10.9 μl dH₂O. This reaction was run on a5% denaturing polyacrylamide gel, and the appropriate region was cut outof the gel. These products were reamplified for an additional 34 cycles,under the conditions listed above, with the exception being that a 42°C. annealing temperature was used.

[0346] Ten μl of the reaction product from run 1 were bound tostreptavidin-coated magnetic beads in 200 μl TE. The beads were washedwith 200 μl TE, and then then resuspended in 20 μl of dH₂O, heatdenatured, and the eluate was removed. Next, 2.5 μl of this eluate wasreamplified for 33 cycles using the conditions indicated above. Thereaction product from run 2 was then added to the beads and diluted with30 μl 0.5× SSC. The mixture was heated from 94° C. to 50° C. The eluatewas removed and the beads were washed three times in 0.5× SSC at 55° C.The beads were then resuspended in 20 μl dH₂O, heat denatured, and theeluate was removed, designated as “round 1 eluate” and saved.

[0347] To isolate the Tetrahymena band, the round 1 eluate wasreamplified with the forward primer K228 (SEQ ID NO:74) and reverseprimer K227 (SEQ ID NO:78) with the sequence5′-CAATTCTC(AG)TA(AG)CA(GATC)(CG)(TA)(CT)TT(AGT)AT(GA)TC-3′ (SEQ IDNO:78), corresponding to the DIKSCYD region (SEQ ID NO:79). The PCRreactions were conducted as described above. The reaction products wererun on a 5% polyacrylamide gel; the band corresponding to approximately295 nucleotides was cut from the gel and sequenced.

[0348] The clone designated as 168-3 was sequenced. The DNA sequence(including the primer sequences) was found to be:GATTACTCCCGAAGAAAGGATCTTTCCGTCCAATCATGACTTTCTTAAGAAAGGACAAGCAAAAAAATATTAAGTTAAATCTAAATTAAATTCTAATGGATAGCCAACTTGTGTTTAGGAATTTAAAAGACATGCTGGGATAAAAGATAGGATACTCAGTCTTTGATAATAAACAAATTTCAGAAAAATTTGCCTAATTCATAGAGAAATGGAAAAATAAAGGAAGACCTCAGCTATATTATGTCACTCTAGACATAAAGA CTTGCTAC (SEQ IDNO:80).

[0349] Additional sequence of this gene was obtained by PCR using oneunique primer designed to match the sequence from 168-3 (“K297” with thesequence 5′-GAGTGACATAATATACGTGA-3′; SEQ ID NO:111), and the K231(FFYXTE) primer. The sequence of the fragment obtained from thisreaction, together with 168-3 is as follows (without the primersequences): AAACACAAGGAAGGAAGTCAAATATTCTATTACCGTAAACCAATATGGAAAT (SEQ IDNO:81). TAGTGAGTAAATTAACTATTGTCAAAGTAAGAATTTAGTTTTCTGAAAAGAATAAATAAATGAAAAATAATTTTTATCAAAAAATTTAGCTTGAAGAGGAGAATTTGGAAAAAGTTGAAGAAAAATTGATACCAGAAGATTCATTTTAGAAATACCCTCAAGGAAAGCTAAGGATTATACCTAAAAAAGGATCTTTCCGTCCAATCATGACTTTCTTAAGAAAGGACAAGCAAAAAAATATTAAGTTAAATCTAAATTAAATTCTAATGGATAGCCAACTTGTGTTTAGGAATTTAAAAGACATGCTGGGATAAAAGATAGGATACTCAGTCTTTGATAATAAACAAATTTCAGAAAAATTTGCCTAATTCATAGAGAAATGGAAAAATAAAGGAAGACCTCAGCTATATTATGTC ACTCTA

[0350] The amino acid sequence corresponding to this DNA fragment wasfound to be: KHKEGSQIFYYRKPIWKLVSKLTIVKVRIQFSEKNKQMKNNFYQKIQLEEENLEKVEEKLIPEDSFQKYPQGKLRIIPKKGSFRPIMTFLRKDKQKNIKLNLNQILMDSQLVFRNLKDMLGQKIGYSVFDNKQISEKFAQFIEKWKNKGRPQLYYVTL (SEQ ID NO:82).

[0351] This amino acid sequence was then aligned with other telomerasegenes (EST2p, and Euplotes). The alignment is shown in FIG. 31.Consensus sequence is also shown in this Figure.

EXAMPLE 16 Identification of Schizosaccharomyces pombe TelomeraseSequences

[0352] In this Example, the tez1 sequence of S. pombe was identified asa homolog of the E. aediculatus p123, and S. cerevisiae Est2p.

[0353]FIG. 33 provides an overall summary of these experiments. In thisFigure, the top portion (Panel A) shows the relationship of twooverlapping genomic clones, and the 5825 bp portion that was sequenced.The region designated at “tez1⁺” is the protein coding region, with theflanking sequences indicated as well, the box underneath the 5825 bpregion is an approximately 2 kb HindIII fragment that was used to maketez1 disruption construct, as described below.

[0354] The bottom half of FIG. 33 (Panel B) is a “close-up” schematic ofthis same region of DNA. The sequence designated as “original PCR” isthe original degenerate PCR fragment that was generated with degenerateoligonucleotide primer pair designed based on Euplotes sequence motif 4(B′) and motif 5 (C), as described in previous Examples.

[0355] PCR With Degenerate Primers

[0356] PCR using degenerate primers was used to find the homolog of theE. aediculatus p123 in S. pombe . FIG. 34 shows the sequences of thedegenerate primers (designated as “poly 4” and “poly 1”) used in thisreaction. The PCR runs were conducted using the same buffer as describedin previous Examples (See e.g., Example 10, above), with a 5 minute ramptime at 94° C., followed by 30 cycles of 94° C. for 30 seconds, 50° C.for 45 seconds, and 72° C. for 30 seconds, and 7 minutes at 72° C.,followed by storage at 4° C. PCR runs were conducted using variedconditions, (i.e., various concentrations of S. pombe DNA and MgCl₂concentrations). The PCR products were run on agarose gels and stainedwith ethidium bromide as described above. Several PCR runs resulted inthe production of three bands (designated as “T,” “M,” and “B”). Thesebands were re-amplified and run on gels using the same conditions asdescribed above. Four bands were observed following thisre-amplification (“T,” “M1,” “M2,” and “B”), as shown in FIG. 35. Thesefour bands were then re-amplified using the same conditions as describedabove. The third band from the top of the lane in FIG. 35 was identifiedas containing the correct sequence for telomerase protein. The PCRproduct designated as M2 was found to show a reasonable match with othertelomerase proteins, as indicated in FIG. 36. In addition to thealignment shown, this Figure also shows the actual sequence of tez1. Inthis Figure, the asterisks indicate residues shared with all foursequences (Oxytricha “Ot”; E. aediculatus “Ea_p123”; S. cerevisiae“Sc_p103”; and M2), while the circles (i.e., dots) indicate similaramino acid residues.

[0357] 3′ RT PCR

[0358] In order to obtain additional sequence information, 3′ and 5′ RTPCR were conducted on the telomerase candidate identified in FIG. 36.FIG. 37 provides a schematic of the 3′ RT PCR strategy used. First, cDNAwas prepared from mRNA using the oligonucleotide primer “Q_(T),” (5′-CCAGTG AGC AGA GTG ACG AGG ACT CGA GCT CAA GCT TTT TTT TTT TTT TT-3′; SEQID NO:102), then using this cDNA as a template for PCR with “Q_(O)′(5′-CCA GTG AGC AGA GTG ACG-3′; SEQ ID NO:103), and a primer designedbased on the original degenerated PCR reaction (i.e., “M2-T” with thesequence 5′-G TGT CAT TTC TAT ATG GAA GAT TTG ATT GAT G-3′ (SEQ IDNO:109). The second PCR reaction (i.e., nested PCR) with “Q_(I)” (5′-GAGGAC TCG AGC TCA AGC-3′; SEQ ID NO:104), and another PCR primer designedwith sequence derived from the original degenerate PCR reaction or“M2-T2” with the sequence 5′-AC CTA TCG TTT ACG AAA AAG AAA GGA TCAGTG-3′; SEQ ID NO:110). The buffers used in this PCR were the same asdescribed above, with amplification conducted beginning with a ramp upof 94° for 5 min, followed by 30 cycles of 94° for 30 sec, 55° C. for 30sec, and 72° C. for 3 min), followed by 7 minutes at 72° C. The reactionproducts were stored at 4° C. until use.

[0359] Screening of Genomic and cDNA Libraries

[0360] After obtaining this extra sequence information, several genomicand cDNA libraries were screened to identify any libraries that containthis telomerase candidate gene. The approach used, as well as thelibraries and results are shown in FIG. 38. In this Figure, Panel Alists the libraries tested in this experiment; Panel B shows the regionsused; Panels C and D show the dot blot hybridization results obtainedwith these libraries. Positive libraries were then screened by colonyhybridization to obtain genomic and cDNA version of tez1 gene. In thisexperiment, approximately 3×10⁴ colonies from the HindIII genomiclibrary were screened and six positive clones were identified(approximately 0.01%). DNA was then prepared from two independent clones(A5 and B2). FIG. 39 shows the results obtained with theHindIII-digested A5 and B2 positive genomic clones.

[0361] In addition, cDNA REP libraries were used. Approximately 3×10⁵colonies were screened, and 5 positive clones were identified (0.002%).DNA was prepared from three independent clones (2-3, 4-1, and 5-20). Inlater experiments, it was determined that 2-3 and 5-20 containedidentical inserts.

[0362] 5′ RT PCR

[0363] As the cDNA version of gene produced to this point was notcomplete, 5′ RT-PCR was conducted in order to obtain a full lengthclone. The strategy is schematically shown in FIG. 40. In thisexperiment, cDNA was prepared using DNA oligonucleotide primer “M2-B”(5′-CAC TGA TCC TTT CTT TTT CGT AAA CGA TAG GT-3′; SEQ ID NO:105) and“M2-B2” (5′-C ATC AAT CAA ATC TTC CAT ATA GAA ATG ACA-3′; SEQ IDNO:106), designed from known regions of tez1 identified previously. Anoligonucleotide linker PCR Adapt SfiI with a phosphorylated 5′ end (“P”)(P-GGG CCG TGT TGG CCT AGT TCT CTG CTC-3′; SEQ ID NO:107) was thenligated at the 3′ end of this cDNA, and this construct was used as thetemplate for nested PCR. In the first round of PCR, PCR Adapt SFI andM2-B were used as the primers; while PCR Adapt SfiII (5-GAG GAG GAG AAGAGC AGA GAA CTA GGC CAA CAC GCC CC-3′; SEQ ID NO:108), and M2-B2 (5′-ATCAAT CAA ATC TTC CAT ATA GAA ATG ACA-3′; SEQ ID NO:106) were used asprimers in the second round. Nested PCR was used to increase specificityof reaction.

[0364] Sequence Alignments

[0365] Once the sequence of tez1 was identified, it was compared withsequences previously described. FIG. 41 shows the alignment of reversetranscriptase (RT) domains from telomerase catalytic subunits of S.pombe (“S.p. Tezlp”), S. cerevisiae (“S.c. Est2p”), and E. aediculatusp123 (“E.a. p123”). In this Figure, “h” indicates hydrophobic residues,while “p” indicates small polar residues, and “c” indicates chargedresidues. The amino acid residues indicated above the alignment showsthe consensus RT motif of Y. Xiong and T. H. Eickbush (Y. Xiong and T.H. Eickbush, EMBO J., 9: 3353-3362 [1990]). The asterisks indicate theresidues that are conserved for all three proteins. “Motif 0” isidentified herein as a motif specific to this telomerase subunit and notfound in reverse transcriptases in general. It is therefore valuable inidentifying other amino acid sequences as being good candidates fortelomerase catalytic subunits.

[0366]FIG. 42 shows the alignment of entire sequences from Euplotes(“Eap123”), S. cerevisiae (“Sc_Est2p”), and S. pombe (“Sp_Tez1p”). InPanel A, the shaded areas indicate residues shared between twosequences. In Panel B, the shaded areas indicate residues shared betweenall three sequences.

[0367] Genetic Disruption of tez1

[0368] In this Example, the effects of disruption of tez1 wereinvestigated. As telomerase is involved in telomere maintenance, it washypothesized that if tez1 were indeed a telomerase component, disruptionof tez1 was expected to cause gradual telomere shortening.

[0369] In these experiments, homologous recombination was used tospecifically disrupt the tez1 gene in S. pombe . This approach isschematically illustrated in FIG. 43. As indicated in FIG. 43, wild typetez1 was replaced with a fragment containing the ura4 or LEU2 marker.

[0370] The disruption of tez1 gene was confirmed by PCR (FIG. 44), andSouthern blot was performed to check for telomere length. FIG. 45 showsthe Southern blot results for this experiment. Because an Apa Irestriction enzyme site is present immediately adjacent to telomericsequence in S. pombe , digestion of S. pombe genomic DNA preparationspermits analysis of telomere length. Thus, DNA from S. pombe wasdigested with ApaI and the digestion products were run on an agarose geland probed with a telomeric sequence-specific probe to determine whetherthe telomeres of disrupted S. pombe cells were shortened. The resultsare shown in FIG. 45. From these results, it was clear that disruptionof the tez1 gene caused a shortening of the telomeres.

EXAMPLE 17 Cloning and Characterization of Human Telomerase Protein andcDNA

[0371] In this Example, the nucleic and amino acid sequence informationfor human telomerase was determined. Partial homologous sequences werefirst identified in a BLAST search conducted using the Euplotes 123 kDapeptide and nucleic acid sequences, as wells as Schizosaccharomycesprotein and corresponding cDNA (tez1) sequences. The human sequences(also referred to as “hTCP1.1”) were identified from a partial cDNAclone (GenBank accession #AA281296). Sequences from this clone werealigned with the sequences determined as described in previous Examples.

[0372]FIG. 25 shows the sequence alignment of the Euplotes (“p123”),Schizosaccharomyces (“tezl”), Est2p (i.e., the S. cerevisiae proteinencoded by the Est2 nucleic acid sequence, and also referred to hereinas “L8543.12”), and the human homolog identified in this comparisonsearch. The amino acid sequence of this aligned portion is provided inSEQ ID NO:61 (the cDNA sequence is provided in SEQ ID NO:62), while theportion of tez1 shown in FIG. 25 is provided in SEQ ID NO:63. Theportion of Est2 shown in this Figure is also provided in SEQ ID NO:64,while the portion of p123 shown is also provided in SEQ ID NO:65. FIG.29 shows the amino acid sequence of tez1 (SEQ ID NO:68), while FIG. 30shows the DNA sequence of tez1 (SEQ ID NO:69). In FIG. 30, the intronsand other non-coding regions, are shown in lower case, while the exons(i.e., coding regions) are shown in upper case.

[0373] As shown in FIG. 25, there are regions that are highly conservedamong these proteins. For example, as shown in this Figure, there areregions of identity in “Motif O,” “Motif 1, ” Motif 2,” and “Motif 3.”The identical amino acids are indicated with an asterisk (*), while thesimilar amino acid residues are indicated by a circle (). Thisindicates that there are regions within the telomerase motifs that areconserved among a wide variety of eukaryotes, ranging from yeast tociliates, to humans. It is contemplated that additional organisms willlikewise contain such conserved regions of sequence. FIG. 27 shows thepartial amino acid sequence of the clone encoding human telomerasemotifs (SEQ ID NO:67), while FIG. 28 shows the corresponding DNAsequence of the Genbank #AA281296 clone.

[0374] Sanger dideoxy sequencing and other methods were used, as knownin the art to obtain complete sequence information of the Genbank clone#AA281296. Some of the primers used in the sequencing are shown in Table3. These primers were designed to hybridize to the clone (GenBankaccession #AA281296), based on sequence complementarity to eitherplasmid backbone sequence or the sequence of the human cDNA insert inthe clone. TABLE 3 Primers Primer Sequence SEQ ID NO: TCP1.1 GTGAAGGCACTGTTCAGCG SEQ ID NO:87 TCP1.2  GTGGATGATTTCTTGTTGG SEQ IDNO:88 TCP1.3  ATGCTCCTGCGTTTGGTGG SEQ ID NO:89 TCP1.4 CTGGACACTCAGCCCTTGG SEQ ID NO:90 TCP1.5  GGCAGGTGTGCTGGACACT SEQ IDNO:91 TCP1.6  TTTGATGATGCTGGCGATG SEQ ID NO:92 TCP1.7 GGGGCTCGTCTTCTACAGG SEQ ID NO:93 TCP1.8  CAGCAGGAGGATCTTGTAG SEQ IDNO:94 TCP1.9  TGACCCCAGGAGTGGCACG SEQ ID NO:95 TCP1.10TCAAGCTGACTCGACACCG SEQ ID NO:96 TCP1.11 CGGCGTGACAGGGCTGC SEQ ID NO;97TCP1.12 GCTGAAGGCTGAGTGTCC SEQ ID NO:98 TCP1.13 TAGTCCATGTTCACAATCG SEQID NO:99

[0375] From these experiments, it was determined that the EcoRI-NotIinsert of the Genbank #AA281296 clone contains only a partial openreading frame for the human telomerase protein, although it may encodean active fragment of that protein. The open reading frame in the cloneencodes an approximately 63 kD protein. The sequence of the longest openreading frame identified is shown in FIG. 47 (SEQ ID NO:100). The ORFbegins at the ATG codon with the “met” indicated in the Figure. The polyA tail at the 3′ end of the sequence is also shown. FIG. 48 shows atentative alignment of telomerase reverse transcriptase proteins fromthe human sequence (human Telomerase Core Protein 1, ” Hs TCP1”), E.aediculatus p123 (“Ep p123), S. pombe tez1 (“Sp Tez1”), S. cerevisiaeEST2 (Sc Est2”), and consensus sequence. In this Figure various motifsare indicated.

[0376] To obtain a full-length clone, probing of a cDNA library and 5′-RACE were used to obtain clones encoding portions of the previouslyuncloned regions. In these experiments, RACE (Rapid Amplification ofcDNA Ends; See e.g., M. A. Frohman, “RACE: Rapid Amplification of cDNAEnds,” in Innis et al. (eds), PCR Protocols: A Guide to Methods andApplications [1990], pp. 28-38; and Frohman et al., Proc. Natl. Acad.Sci., 85:8998-9002 [1988]) was used to generate material for sequenceanalysis. Four such clones were generated and used to provide additional5′ sequence information (pFWRP5, 6, 19, and 20).

[0377] In addition, human cDNA libraries (inserted into lambda) wereprobed with the EcoRI-NotI fragment of the clone (#AA281296). One lambdaclone, designated “lambda 25-1.1,” (ATCC accession #______) wasidentified as containing complementary sequences. FIG. 54 shows arestriction map of this lambda clone. The human cDNA insert from thisclone was subcloned as an EcoRI restriction fragment into the EcoRI siteof commercially available phagemid pBluescriptIISK+ (Stratagene), tocreate the plasmid “pGRN121,” which was deposited with the ATCC (ATCCaccession #209016). Preliminary results indicated that plasmid pGRN121contains the entire open reading frame (ORF) sequence encoding the humantelomerase protein.

[0378] The cDNA insert of plasmid pGRN121 was sequenced using techniquesknown in the art. FIG. 49 provides a restriction site and function mapof plasmid pGRN121 identified based on this preliminary work. Theresults of this preliminary sequence analysis are shown in FIG. 50. Fromthis analysis, and as shown in FIG. 49, a putative start site for thecoding region was identified at approximately 50 nucleotides from theEcoRI site (located at position 707), and the location of thetelomerase-specific motifs, “FFYVTE” (SEQ ID NO:1 12), “PKP,” “AYD,”“QG”, and “DD,” were identified, in addition to a putative stop site atnucleotide #3571 (See, FIG. 51). FIG. 51 shows the DNA and correspondingamino acid sequences for the open reading frames in the sequence (“a”[SEQ ID NO:114], “b” [SEQ ID NO:115], and “c” [SEQ ID NO:116]). However,due to the preliminary nature of the early sequencing work, the readingframes for the various motifs were found not to be in alignment.

[0379] Additional analysis conducted on the pGRN121 indicated that theplasmid contained significant portions from the 5′-end of the codingsequence not present on the Genbank accession #AA281296 clone.Furthermore, pGRN121 was found to contain a variant coding sequence thatincludes an insert of approximately 182 nucleotides. This insert wasfound to be absent from the Genbank accession #AA281296 clone. As withthe E. aediculatus sequences, such variants can be tested in functionalassays, such as telomerase assays to detect the presence of functionaltelomerase in a sample.

[0380] Further sequence analysis resolved the cDNA sequence of pGRN121,to provide a contiguous open reading frame that encodes a protein ofmolecular weight of approximately 127,000 daltons, and 1132 amino acidsas shown in FIG. 53 (SEQ ID NOS:117 and 118). A refined map of pGRN121based on this analysis, is provided in FIG. 52.

[0381] From the above, it is clear that the present invention providesnucleic acid and amino acid sequences, as well as other informationregarding telomerase, telomerase protein subunits, and motifs fromvarious organisms, in addition to methods for identification ofhomologous structures in other organisms in addition to those describedherein.

[0382] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

We claim:
 1. A substantially purified peptide comprising the amino acidsequence selected from the group consisting of SEQ ID NOS:71, 73, 75,77, 79, 82, 83, 83, 85, 86, and
 101. 2. A purified, isolatedpolynucleotide sequence encoding the polypeptide of claim
 1. 3. Thepolynucleotide sequence of claim 2, wherein said polynucleotidehybridizes specifically to telomerase sequences, wherein said telomerasesequences are selected from the group consisting of human, Euplotesaediculatus, Oxytricha, Schizosaccharomyces, and Saccharomycestelomerase sequences
 4. The polynucleotide sequence of claim 3,comprising the complement of a nucleic acid sequence selected from thegroup consisting of SEQ ID NOS:70, 72, 74, 76, 78, 80, 81, and 100, andvariants thereof.
 5. A polynucleotide sequence that hybridizes understringent conditions to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS:66, 69, 80, and
 81. 6. The polynucleotidesequence of claim 5, wherein said polynucleotide sequence is selectedfrom the group consisting of SEQ ID NOS:70, 72, 74, 76, 78, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 102, 103, 104, 105, 106, 107,108, 109, and
 110. 7. The polynucleotide sequence of claim 6, whereinsaid nucleotide sequence comprises a purified, synthetic nucleotidesequence having a length of about ten to fifty nucleotides.
 8. A methodfor detecting the presence of polynucleotide sequences encoding at leasta portion of human telomerase in a biological sample, comprising thesteps of: a) providing: i) a biological sample suspected of containingnucleic acid corresponding to the polynucleotide sequence of SEQ IDNO:100; ii) the nucleotide sequence of SEQ ID NO:100, or a fragmentthereof; b) combining said biological sample with said nucleotide underconditions such that a hybridization complex is formed between saidnucleic acid and said nucleotide; and c) detecting said hybridizationcomplex.
 9. The method of claim 8, wherein, said nucleic acidcorresponding to the nucleotide sequence of SEQ ID NO:100 is ribonucleicacid.
 10. The method of claim 9, wherein said detected hybridizationcomplex correlates with expression of the polynucleotide of SEQ ID NO:100 in said biological sample.
 11. The method of claim 8, wherein, saidnucleic acid corresponding to the nucleotide sequence of SEQ ID NO:100is deoxyribonucleic acid.
 12. The method of claim 11, wherein saiddetecting of said hybridization complex comprises conditions that permitthe detection of alterations in the nucleotide of SEQ ID NO:100 in saidbiological sample.
 13. An antisense molecule comprising the nucleic acidsequence complementary to at least a portion of the nucleotide of SEQ IDNO:
 100. 14. A pharmaceutical composition comprising the antisensemolecule of claim 13, and a pharmaceutically acceptable excipient. 15.The polynucleotide sequence of claim 4, wherein said nucleotide sequenceis contained on a recombinant expression vector.
 16. The polynucleotidesequence of claim 15, wherein said expression vector containing saidnucleotide sequence is contained within a host cell.
 17. A method forproducing a polypeptide comprising the amino acid sequence of SEQ IDNO:101, the method comprising the steps of: a) culturing the host cellof claim 16, under conditions suitable for the expression of thepolypeptide; and b) recovering the polypeptide from the host cellculture.
 18. A purified antibody which binds specifically to apolypeptide comprising at least a portion of the amino acid sequence ofSEQ ID NO:101.
 19. A pharmaceutical composition comprising the antibodyof claim 18 and a pharmaceutically acceptable excipient.
 20. A methodfor detecting the expression of human telomerase in a biological samplecomprising the steps of: a) providing: i) a biological sample suspectedof expressing human telomerase protein; and ii) the antibody of claim18; b) combining said biological sample and said antibody underconditions such that an antibody:protein complex is formed; and c)detecting said complex wherein the presence of said complex correlateswith the expression of said protein in said biological sample.